D7. Redox Chemistry and Protein Folding - Biology

D7. Redox Chemistry and Protein Folding - Biology

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In general we envision the interior of a cell to be in a reducing environment. Cells have sufficient concentrations of "b-mercaptoethanol"-like molecules (used to reduce disulfide bonds in proteins in vitro) such as glutathione (g-Glu-Cys-Gly) and reduced thioredoxin (with an active site Cys) to prevent disulfide bond formation in cytoplasmic proteins. Most cytoplasmic proteins contain Cys with side chain pKa > 8, which would minimize disulfide bond formation as the Cys are predominantly protonated at that pH.

Disulfide bonds in proteins are typically found in extracellular proteins, where they serve to keep multisubunit proteins together as they become diluted in the extracellular milieu. These proteins destined for secretion are cotranslationally inserted into the endoplasmic reticulum (see below) which presents an oxidizing environment to the folding protein and where sugars are covalently attached to the folding protein and disulfide bonds are formed (see Chapter 3D: Glycoproteins - Biosynthesis and Function). Protein enzymes involved in disulfide bond formation contain free Cys which form mixed disulfides with their target substrate proteins. The enzymes (thiol-disulfide oxidoreductases, protein disulfide isomerases) have a Cys-XY-Cys motif and can promote disulfide bond formation or their reduction to free sulfhydryls. They are especially redox sensitive since their Cys side chains must cycle between and free disulfide forms.

Intracellular disulfide bonds are found in protein in the periplasm of prokaryotes and in the endoplasmic reticulum (ER) and mitochondrial intermembrane space (IMS) of eukaryotes. For these proteins, the beginning stage of protein synthesis (in the cytoplasm) is separated temporally and spatially from the site of disulfide bond formation and final folding. Disulfide bonds can be generated in a target protein by concomitant reduction of a disulfide in a protein catalyst, leaving the net number of disulfides constant (unless the enzyme is reoxidized by an independent process). Alternatively, a disulfide can be formed by transfer of electrons to oxidizing agents such as dioxygen.

In the ER, disulfide bond formation is catalyzed by proteins in the disulfide isomerase family (PDI). To function as catalysts in this process, the PDIs must be in an oxidized state capable of accepting electrons from the protein target for disulfide bond formation. A flavoprotein, Ero1, recycles PDI back to an oxidized state, and the reduced Ero1 is regenerated on passing electrons to dioxygen to form hydrogen peroxide. In summary, on formation of disulfides in the ER, electrons flow from the nascent protein to PDIs to the flavin protein Ero1 to dioxgen (i.e. to better and better electron acceptors). The first step is really a disulfide shuffle, which, when coupled to the subsequent steps, leads to de novo disulfide bond formation.

In the mitochondria, disulfide bond formation occurs in the intermembrane space (IMS) and is guided by the �mitochondria disulfide relay system.� This system requires two important proteins: Mia40 and Erv1. Mia40 contains a redox active disulfide bond cys-pro-cys and oxidizes cys residues in polypeptide chains. Erv1 can then reoxidize Mia40 which can in turn get reoxized itself by the heme in cytochrome c. Reduced cytochrome C is oxidized by cytochrome C oxidase of electron transport through passage of electrons to dioxygen to form water. The importance of IMS protein oxidation is less understood, but it is believe that the oxidative stress caused by a dysfunction could lead to neurodegenerative diseases.

A recent review by Riemer et al compares the ER and mitochondrial processes for disulfide bond formation:

  • Many more and diverse proteins form disulfides in the ER compared to the IMS. Most in the IMS have low molecular mass and have two disulfide bonds between helix-turn-helix motifs. These protein substrates include chaperones that facilitate localization of proteins in the inner membrane, and in proteins involved in electron transport in the inner membrane.

  • There are many PDIs in the ER, probably reflecting the structural diversity of protein substrates in the ER. However Mia40 appears to be the only PDI in the IMS.

  • "De novo" disulfide bond formation is initiated by Ero1 in the ER and Erv1 in the IMS. Convergent evolution led to the similar structures for both - a 4-helix bundle that binds FAD with two proximal Cys.

  • The mitochondria pathway lead to water formation on reduction of dioxygen, not hydrogen peroxide, minimizing the formation of reactive oxygen species in the mitochondria. The peroxide formed in the ER is presumably convert to an inert form.

  • The IMS is in more intimate contact with the cytoplasm through outer membrane proteins called porins which would allow some glutathione access. The IMS presents a more oxidizing environment than the cytoplasm (with more glutathione). The ER, without a porin analog, would be more oxidizing.

  • Reversible formation of disulfides in the ER regulates protein activity.

Disulfide bond regulation in the Periplasmic Space of Bacteria

The redox sensitivity of the Cys side chain found in disulfide bonds is important in regulating protein activity. In particular, the thiol group of the amino acid Cys, an important nucleophile often found in active site, can be modified to control protein activity. The formation of a disulfide bond or the oxidation of free thiols to sulfenic acid or further to sulfinic or sulfonic acid can block protein activity. The E. Coli periplasmic proteins DsbA (disulfide bond A) converts adjacent free thiols into disulfide-linked Cystine, in the process becoming reduced. DsbB reoxidized DsbA back to its catalytyically active form. What about periplasmic protein like YbiS with an active site Cys? Since the environment of the periplasm is oxidizing, YbiS mist be protected from oxidative conversion of the free Cys to either sulfinic or sulfonic acids causing the protein to become inactive. The mechanism involves two periplasmic proteins known as DsbG and DsbC which are similar to thioredoxin. These two proteins are able to donate electrons to the unprotected thiol preventing it from becoming oxidized, which allows YbiS to remain active in the periplasm. To maintain activity, DsbG and DsbC are reduced by another periplasmic protein, DsbD.

Protein folding and disulfide bond formation in the eukaryotic cell: meeting report based on the presentations at the European Network Meeting on Protein Folding and Disulfide Bond Formation 2009 (Elsinore, Denmark)

The endoplasmic reticulum (ER) plays a critical role as a compartment for protein folding in eukaryotic cells. Defects in protein folding contribute to a growing list of diseases, and advances in our understanding of the molecular details of protein folding are helping to provide more efficient ways of producing recombinant proteins for industrial and medicinal use. Moreover, research performed in recent years has shown the importance of the ER as a signalling compartment that contributes to overall cellular homeostasis. Hamlet's castle provided a stunning backdrop for the latest European network meeting to discuss this subject matter in Elsinore, Denmark, from 3 to 5 June 2009. Organized by researchers at the Department of Biology, University of Copenhagen, the meeting featured 20 talks by both established names and younger scientists, focusing on topics such as oxidative protein folding and maturation (in particular in the ER, but also in other compartments), cellular redox regulation, ER-associated degradation, and the unfolded protein response. Exciting new advances were presented, and the intimate setting with about 50 participants provided an excellent opportunity to discuss current key questions in the field.

Thiol Redox Transitions in Cell Signaling, Part A: Chemistry and Biochemistry of Low Molecular Weight and Protein Thiols

Pierluigi Mauri , . Fulvio Ursini , in Methods in Enzymology , 2010

2 MS/MS Identification of Redox-Switches in Protein

In protein chemistry , structural disulfides are identified by comparing and resequencing peptides produced by different endoproteases under reducing and nonreducing conditions. These approaches (reviewed in Gorman et al., 2002 ) are long and complex and today are progressively substituted by modern MS/MS-based technology.

When a protein containing a redox-switch is in its oxidized form, proteolysis produces either a fragment containing the disulfide or double fragments linked to each other by the disulfide. In both cases, the appearance of a new peptide with a mass 2 a.m.u. lower than the mass of either the reduced peptide or the sum of two peptides suggests an identification that will be validated by MS/MS sequencing.

The major drawback of the approach is the need of an alkaline pH for the activity of trypsin, the most specific and standardized endoprotease used in proteomics. In fact, when Cys are dissociated, a thiol–disulfide scrambling takes place by a nucleophilic attack of thiolate on the disulfide, as already pointed out by Sanger in 1953 . This artifactual reshuffling deeply weakens the validity of the disulfide assignment ( Gorman et al., 2002 ).

Although this major pitfall is often overlooked in reports of disulfide identification, including glutathionylation, its occurrence, when trypsin is used and the protein contains free cysteines, is unavoidable. The use of alkylating agents to scavenge free Cys in our hands was not satisfactory since the possibility that a thiolate still reacts with a disulfide instead of reacting with the alkylating agent, cannot be fully ruled out.

The use of the endoprotease pepsin, which is active at acidic pH, prevents the thiol–disulfide scrambling, but this approach suffers from the low and unpredictable specificity of the proteolysis.

In our studies, this problem was worked out by heuristically assuming a constant proteolytic pattern in the oxidized and reduced protein. Candidate double peptides were identified according to the pattern of fragmentation observed under reducing conditions. MS/MS analysis of the double b-y series confirmed the correct identifications.

The oxidase DsbA folds a protein with a nonconsecutive disulfide

One of the last unsolved problems of molecular biology is how the sequential amino acid information leads to a functional protein. Correct disulfide formation within a protein is hereby essential. We present periplasmic ribonuclease I (RNase I) from Escherichia coli as a new endogenous substrate for the study of oxidative protein folding. One of its four disulfides is between nonconsecutive cysteines. In general view, the folding of proteins with nonconsecutive disulfides requires the protein disulfide isomerase DsbC. In contrast, our study with RNase I shows that DsbA is a sufficient catalyst for correct disulfide formation in vivo and in vitro. DsbA is therefore more specific than generally assumed. Further, we show that the redox potential of the periplasm depends on the presence of glutathione and the Dsb proteins to maintain it at-165 mV. We determined the influence of this redox potential on the folding of RNase I. Under the more oxidizing conditions of dsb(-) strains, DsbC becomes necessary to correct non-native disulfides, but it cannot substitute for DsbA. Altogether, DsbA folds a protein with a nonconsecutive disulfide as long as no incorrect disulfides are formed.

Evidence for redox sensing by a human cardiac calcium channel

Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses. Calcium influx into heart muscle occurs through the ion conducting α1C subunit (Cav1.2) of the L-type Ca(2+) channel. Glutathionylation of Cav1.2 results in increased calcium influx and is evident in ischemic human heart. However controversy exists as to whether direct modification of Cav1.2 is responsible for altered function. We directly assessed the function of purified human Cav1.2 in proteoliposomes. Truncation of the C terminus and mutation of cysteines in the N terminal region and cytoplasmic loop III-IV linker did not alter the effects of thiol modifying agents on open probability of the channel. However mutation of cysteines in cytoplasmic loop I-II linker altered open probability and protein folding assessed by thermal shift assay. We find that C543 confers sensitivity of Cav1.2 to oxidative stress and is sufficient to modify channel function and posttranslational folding. Our data provide direct evidence for the calcium channel as a redox sensor that facilitates rapid physiological responses.


Figure 1. Characterisation of the Ca v…

Figure 1. Characterisation of the Ca v 1.2 current of the human long NT isoform…

Figure 2. The long NT isoform and…

Figure 2. The long NT isoform and short NT isoform respond similarly to DTT and…

Figure 3. Truncating the C terminus of…

Figure 3. Truncating the C terminus of the long NT isoform significantly increases P o…

Figure 4. Mutation of cysteines in the…

Figure 4. Mutation of cysteines in the N-terminus of the long NT isoform do not…

Figure 5. Mutation of cysteines in the…

Figure 5. Mutation of cysteines in the cytoplasmic loop III-IV linker of the long NT…

Figure 6. Mutation of cysteines in the…

Figure 6. Mutation of cysteines in the loop I-II linker region of the long NT…

Figure 7. Mutation of cysteine 543 to…

Figure 7. Mutation of cysteine 543 to serine alters the response of the channel to…

Structure-function relations, physiological roles, and evolution of mammalian ER-resident selenoproteins

Selenium is an essential trace element in mammals. The major biological form of this micronutrient is the amino acid selenocysteine, which is present in the active sites of selenoenzymes. Seven of 25 mammalian selenoproteins have been identified as residents of the endoplasmic reticulum, including the 15-kDa selenoprotein, type 2 iodothyronine deiodinase and selenoproteins K, M, N, S, and T. Most of these proteins are poorly characterized. However, recent studies implicate some of them in quality control of protein folding in the ER, retrotranslocation of misfolded proteins from the ER to the cytosol, metabolism of the thyroid hormone, and regulation of calcium homeostasis. In addition, some of these proteins are involved in regulation of glucose metabolism and inflammation. This review discusses evolution and structure-function relations of the ER-resident selenoproteins and summarizes recent findings on these proteins, which reveal the emerging important role of selenium and selenoproteins in ER function.


Natural linkers act as spacers between the domains of multidomain proteins. Several interdomain linkers have been identified at the boundaries of functionally distinct domains. 1 These linkers adopt a coiled structure and show a preference for Gln, Arg, Glu, Ser, and Pro amino acids. 1 An automated method to extract interdomain linkers from a dataset of proteins with a known three-dimensional structure revealed that the majority of the interdomain linker sets constitutes Pro, Arg, Phe, Thr, Glu, and Gln residues that act as rigid spacers. 2 Proline is common to many naturally derived interdomain linkers, and structural studies indicate that proline-rich sequences form relatively rigid extended structures to prevent unfavorable interactions between the domains. 3 An independent analysis showed that Thr, Ser, Gly, and Ala are also preferred residues in natural linkers. 4 Besides, flexible Gly-rich regions have been observed as natural linkers in proteins, generating loops that connect domains in multidomain proteins. 5 , 6 The advancement in recombinant DNA technology facilitated fusion of proteins or domains using these synthetic amino acid linkers. In addition to structural studies of protein–protein interactions, a wide range of applications in the field of biotechnology have employed these fused proteins to explore protein-based biochemistry, such as to create artificial bifunctional enzymes, 7 to produce antibodies 8 and proteins with specialized functions, 9 and as tools for FRET analysis. 10

The characterization of protein–protein interactions is often required to gain an understanding of various biological processes. Yet, the study of protein–protein interactions for many complexes is hampered when one or more partners of the complex are unfolded or unstable. Traditionally, this problem has been addressed by the co-expression and/or co-purification of both proteins. 11 However, for weakly interacting or unstable complexes, the co-expression and/or co-purification often results in a single protein. 12 Protein engineering techniques were another option to address unfolded or unstable proteins, using a single polypeptide chain chimera to link the two binding partners via a flexible amino acid linker 13 . With these chimeric proteins, it was then possible to maintain both the intramolecular and intermolecular protein–protein interactions, 14 and chimeric proteins have been used to generate stable, soluble binary complexes for structural studies, as well as functional dimers. 15

Linking binding partners using an artificial linker will increase the proximity between the interacting partners and preserve the natural interaction. In cases where the interacting partners are not linked, it is possible that the binding partners might dissociate due to their low affinity and/or due to the crystallization conditions. Most of the artificial linkers used to generate chimeric proteins for structural studies are rich in Gly residues (Table I). Interestingly, the naturally occurring, flexible linkers found in many proteins are also rich in Gly residues. In the next section, we discuss some of these naturally occurring Gly-rich linkers.

1FTK, 1FTL, 1FTO, 1FW0, 1FTJ, 1FTM

Gly-rich linkers in nature

Naturally occurring Gly-rich linkers exist in many proteins and, aside from linking domains, they are known to have a functional role in the protein. The transcription factor PAX6 consists of two DNA-binding domains, a paired domain (PD) and a homeodomain (HD), joined by a Gly-rich linker. 16 Crystal structure analysis of the human PAX6 PD-DNA complex revealed that the extended linker makes minor groove contacts with the DNA. 17 In transmembrane glycoproteins (TMs) of retroviruses, important functional roles are also carried out by the linkers, which mediate membrane fusion through an N-terminal fusion peptide. The fusion peptide is linked to the central coiled-coil core through Gly-rich linkers. The length and amino acid composition of linkers are conserved in many retroviruses and in HA2 of influenza viruses. 18 Alanine and proline scanning mutagenesis experiments showed that the Gly-rich linker of human T-cell leukemia virus Type 1 (HTLV-1) TM, gp21, is involved in membrane fusion function. 18 The N-terminus of the SR protein (sequence-specific RNA-binding factors), SRSF1, contains two RNA recognition motifs (RRM) connected through a Gly-rich linker, which is essential for the binding of exonic splicing enhancers (ESE). 19 In other cases, the role of the Gly-rich linker is still unknown. In filamentous Ff bacteriophages (M13, fd, and f1) minor coat gene 3 protein (g3p) consists of three domains (N1, N2, and CT) connected by a flexible Gly-rich linker 6 a longer Gly-rich region exists in the g3p of the IKe filamentous phage, but this appears to confer no selective advantage. 20 Gly-rich linkers are also involved in disease conditions. TDP-43 (TAR DNA binding protein) consists of two RNA binding domains (RBD) and a Gly-rich C-terminus. Mutations in the Gly-rich domain of TDP-43 have been related to amyotrophic lateral sclerosis. 21 Gly-rich linkers are also employed for bioimaging studies. Novel fluorogen activating proteins (FAPs) consist of single-chain variable fragments (scFvs), which are made of human immunoglobulin variable heavy (VH) and variable light (VL) domains covalently attached via a Gly- and Ser-rich linker. 22 These FAPs produce fluorescence upon binding to a specific dye or fluorogen and are used in bio-imaging studies to track protein dynamics.

The roles of linkers

Poly-Gly and Gly-rich linkers can be considered as independent units and do not affect the function of the individual proteins to which they attach. The fused proteins behave independently, such that the single chained proteins can perform the combined function of fused partners. 23 , 24 For example, the native Type I' beta-turn loop in staphylococcal nuclease was replaced with a five-residue turn sequence from concanavalin A, without altering the activity of the nuclease. The crystal structure revealed that the hybrid protein was well folded and the introduced turn sequence retained the conformation of the parent concanavalin A structure. 25 In other systems, however, linker regions can affect the stability, solubility, oligomeric state, and proteolytic resistance of the fused proteins. 24 The repressor protein, Arc, is a dimer with identical subunits. A fused protein—Arc-linker-Arc—was tested for its stability, protein folding kinetics, and biological activity by varying the length and composition of the linker. While the poly-Gly linkers provided maximum conformational freedom, it failed to ensure optimal stability. In contrast, maximum stability was obtained with a linker containing 11 Ala and 5 Gly residues or 7 Ser and 9 Gly residues, respectively, in the randomized region of the linker. 26 Flexible Gly linkers have been used to improve the folding and function of epitope-tagged proteins. Flexible polypeptide linkers of 5, 8, or 10 Gly residues have been placed between an epitope and a tagged protein to increase epitope sensitivity and accessibility without interfering in protein folding and function. 27 , 28 Thus, it is important that the length and amino acid composition of a potential linker is optimized in order to preserve the biological activity of the individual proteins in the fused complex.

The loop length created by the linker can have a profound effect on the action of the linker in the fused complex. A systematic study to investigate the role of loop length in protein folding and stability was carried out using the Rop protein as a model system. 24 Rop is a homodimer of helix-loop-helix monomers and, for this experiment, the two-residue loop linker between helix 1 and helix 2 was replaced by a series of non-natural (Gly)1–10 linkers, with 1 to 10 Gly residues. As with the wild type Rop protein, all 10 mutants maintained a highly helical dimer structure and retained the same level of wild-type RNA-binding activity. Interestingly, however, the stability of the Rop protein toward thermal and chemical denaturation was inversely correlated with loop length that is, a longer loop length resulted in a progressively decreased stability. 24 In cases where protein solubility and/or stability are the key problems for protein–protein interaction studies, tethering them to each other can improve the solubility and stability of the complex. 30 , 31 In some cases, the stability can be improved by altering the linker length and amino acid composition. 26 Thus, Gly-rich linkers are used between two interacting proteins either to improve the stability of one of the binding partners or, where the interaction is transient, to generate a stable protein–protein complex. Here, we present our analysis on various cases where artificial Gly-rich linkers have been used to fuse two proteins for structural studies. Table I provides details about the proteins that were fused and also about the length and composition of the linker used for fusion.

Linkers for unstable proteins and their binding partners

Some proteins exist in complex with their binding partner(s) in order to remain stable in cells. 23 They cannot exist as a single protein and require co-expression with their binding partner when expressed in a heterologous system. Overexpression of single proteins would result in unstable protein molecules or higher molecular weight aggregates, which hampers crystallization. In several cases, this is addressed via the use of flexible Gly-rich linkers. In the following sections, we briefly discuss the utilization of this strategy to generate stable major histocompatibility complex (MHC) Class II molecules, LIM (Lin-11/Islet-1/Mec-3) domains and Pregnane X receptor molecules for structural studies.

MHC class II-peptide complexes

The linkers used in MHC molecules are advantageous for two different scenarios: to stabilize proteins and to trap transient protein–protein interactions. Initially, it was shown that the covalent linkage of a peptide to an MHC Class II molecule could stabilize the MHC molecule. 13 Later, this strategy was applied to obtain the MHC/peptide/TCR (T-cell receptor) ternary complex, 75 which was revealed to be a fast dissociating complex (see Linkers to trap transient protein–protein interactions section).

MHC Class II molecules are heterodimeric proteins comprising noncovalently interacting α and β chains. The extracellular portion consists of two regions: a peptide binding site, consisting of an α1 or β1 domain, and an Ig-like domain, consisting of an α2 or β2 domain. When expressed as two different chains (α and β) and mixed together to form a αβ heterodimer, MHC Class II molecules resulted in high molecular weight aggregates, suggesting the possible heterogeneity of αβ heterodimers in the presence of free α and β chains. However, stable αβ heterodimers were produced when an antigenic peptide, recognized by a particular MHC Class II molecule, was covalently linked to the N-terminus of the MHC Class II β1 chain by a 16-amino acid Gly-rich linker (GGGGSLVPRGSGGGGS). The presence of linker had no effect on the recognition of peptide-MHC Class II complex by the specific T-cell hybridomas. 13 This particular linker has a thrombin cleavage site flanked by Gly-rich residues, so that the linker between the fused proteins can be cleaved. However, the crystal structure of the human class II MHC protein, HLA-DR1, complexed with an unlinked influenza virus peptide (PDB: 1DLH) revealed that a six-amino acid linker is sufficient to connect the C-terminus of the peptide to the N-terminus of the MHC class II molecule 76 . Further, tethering an ovalbumin (OVA) peptide to the N-terminus of the β1 chain of the I-A d MHC Class II molecule using a six-amino acid linker (Table I), was sufficient to enhance the stability of αβ heterodimers (Fig. 1). 78 Further crystallization trials found that many conditions could produce diffraction-quality crystals. This experimental approach led to the successful crystallization of other MHC Class II-peptide complexes using a polypeptide linker. 31-44

Crystal structure of MHC Class II-peptide complex. Ribbon representation of MHC Class II I-A d (green) and ovalbumin peptide (magenta) complex (PDB code: 2IAD), where the C-terminus of ovalbumin peptide is linked to the N-terminus of the β chain of MHC Class II I-A d molecule using a GSGSGS (6aa) linker. The electron density for the linker was not observed and the possible position was indicated as blue dotted lines. All structure-related figures of this review were prepared using the program PyMol. 77

LIM domain and related proteins

LIM (Lin-11/Islet-1/Mec-3) domains are a class of zinc binding domains that mediate protein–protein interactions to regulate cell fate. 23 LIM domain-containing proteins are divided into three groups: LIM-only (LMO), LIM homeodomain (LIM-HD) proteins, and LIM kinase families. Generally, LMO proteins are localized in the nucleus, but lack a nuclear localization sequence. Thus, to keep these proteins in the nucleus they must be bound to LIM domain binding protein-1 ( ldb1). LMO1, 2, and 4 play a major role in normal and leukemic T-cell development, and the LMO- ldb1 complexes may modulate gene expression. 79 LMO proteins are insoluble and unstable when expressed by bacteria but this can be rectified when they are fused with ldb1 using either a GGSGGHMGSGG or a GGSGGSGGSGG linker at the C-terminus. 80 The LIM interaction domain (LID) of ldb1 (300–340) is unstructured in solution. 29 Upon binding to the N-terminal LIM domains of LMO2 and LMO4, the LID of ldb1 adopts an extended conformation, stretched along the face of the LIM domain [Fig. 2(A)] to facilitate binding.

Structural details of LIM domains and their binding partner complexes. (A) Ribbon representation of the crystal structure of LMO4 (green)-ldb1 (magenta) complex (PDB code: 1RUT), where the C-terminus of LMO4 is linked to the N-terminus of Ldb1 using a GGSGGSGGSGG (11aa) linker. The blue dotted line represents the possible position of the missing amino acids of the linker. (B) NMR structure of Lhx3 (green)-ldb1 (magenta) complex (PDB code: 2JTN), where the C-terminus of ldb1 is linked to N-terminus of Lhx3 using a GGSGGHMGSGG (11aa) linker (dark blue). Zn 2+ ions are shown as orange spheres.

Likewise, LIM-HD proteins tend to be insoluble and unstable. There are 12 mammalian LIM homeodomain proteins consisting of six pairs of paralogues, which often have overlapping expression patterns and functions. Two such paralogous pairs are LIM homeobox protein 3 and 4 (Lhx3 and Lhx4) and Islet-1 and -2 (Isl1 and Isl2). When expressed in bacteria, the LIM domains from Lhx3 and Lhx4 tend to aggregate and are largely insoluble. Linking an interaction partner, such as Isl1 or ldb1, to the LIM domains can prevent this aggregation and allow production of soluble tethered complexes 81 [Fig. 2(B)]. Since then, various LIM domain structures (Table I) have been solved by exploiting the same technique. 23 , 29 , 30 , 45-50 Figure 2(A,B) shows the interaction between lbd1 and the different LIM domains. These structures were solved using X-ray and NMR techniques. The backbone assignment by NMR experiments for the Gly-rich linker revealed that the linker exists as a random coil. 47

PXR-LBD and SRC-1 complex

Pregnane X receptor (PXR) is a xenobiotic receptor and a ligand-inducible transcription factor involved in regulating drug metabolizing enzymes and transporters. PXR contains two domains: a highly conserved DNA-binding domain (DBD) at the N-terminus and a more divergent ligand-binding domain (LBD) at the C-terminus. The LBD of PXR is involved in ligand recognition and also interacts with co-activator proteins. PXR is an unstable protein and exists as a complex with its co-repressor. Ligand binding displaces the co-repressor by a co-activator, the steroid receptor activator 1 (SRC-1). Studying the interaction between the PXR-LBD and SRC-1 is important in order to understand the mechanism of ligand-mediated transcriptional effects. A dual expression system was initially used to characterize this binding, but resulted in the production of unstable proteins and a stoichiometry of SRC-1 to PXR of less than 1:1. Watkins et al. purified PXR using the SRC-1 domain and then combined this PXR with a SRC-1 interacting peptide to form a complex and determine the crystal structure (PDB: 1NRL). 82 They found that the C-terminus of PXR lies near the N-terminus of SRC-1, at a distance of 18 Å, which would require at least five amino acids to link these proteins. Thus, the SRC-1 peptide was fused to the C-terminus of the PXR-LBD using various length linkers such as S GGG SSHS, S GGSGG SSHS, and S GGSGGSGG SSHS (underlined sequences are the added linker the flanking regions are from the interacting proteins), with most crystals obtained with the 10-amino acid Gly-rich linker 51 (Fig. 3).

Crystal structure of the ligand binding domain of Pregnane X receptor (PXR-LBD) and steroid receptor activator 1 (SRC-1). Ribbon representation of PXR-LBD (green)—SRC-1 interacting peptide (magenta) complex, where the C-terminal of PXR-LBD is linked using a GGSGG (5aa) linker to the N-terminus of the SRC-1 interacting peptide (PDB code: 3CTB). The blue dotted line shows the possible positions of the missing amino acids of the linker.

Linkers to trap transient protein–protein interactions

Several key biological events are dictated by protein–protein interactions, most of which are transient and weak. Trapping a weak or transient interaction for structural studies is always challenging, and flexible Gly-rich linkers have thus proven helpful in these instances for example, trapping of fast dissociating reactions, such as the MHC molecule-bound peptide recognition by TCR the interaction between a phosphoprotein and a nucleocapsid protein of the paramyxovirus as well as trapping low-affinity binding interactions, such as in the case of g3p-TolA and Histone H3-Asf1 (Anti-silencing function 1). Further, in the case of calmodulin (CaM) and the calmodulin binding domain (CBD) of calcineurin, this strategy has been utilized to trap an unstructured protein that gains secondary structure upon binding with its partner. This wide usage of linkers is briefly discussed in the following sections.

T-cell receptor, peptide and MHC class II complexes

T-cell receptors (TCR) are found on the surface of T lymphocytes and recognize antigens bound to major histocompatibility complex (MHC) molecules. When the TCR engages with an antigen and MHC, it becomes activated to enhance the immune response. However, studying these interactions has proven difficult in the past due to a very low affinity between the TCR and the peptide/MHC molecule. Kinetic studies also indicated a slow association and fast dissociation, which made it difficult to trap this transient interaction for further studies. 75 , 83 Hence, to stabilize the TCR/peptide/MHC complex for crystallization, the antigen peptide was covalently linked using a Gly-rich linker at the N-terminus of the MHC molecule β chain (see also MHC class II-peptide complexes section) this linker helped to mediate the formation of a stable TCR-antigen peptide-MHC molecule ternary complex. 52-54 Another way to stabilize this complex was shown using an eight-amino acid polypeptide (GGSGGGGG) to link the antigen peptide and the N-terminus of the variable β chain of TCR. 55-58 The overall structure of the TCR-peptide-MHC and the MHC-peptide-TCR molecules did not show any differences in the binding regions of MHC and TCR molecules using both methods [Fig. 4(A,B)] thus, the structure was able to be solved as a ternary complex. Table I outlines the various TCR-peptide-MHC ternary complexes that were solved using these methods.

Crystal structures of different MHC molecule-peptide-T cell receptor (TCR) complexes. (A) Ribbon representation of TCR D10 (green)-hen egg CA peptide (yellow)-MHC Class II I-A k (magenta) complex (PDB code: 1D9K), where the C-terminus of the CA peptide is linked to the N-terminus of the β chain of the MHC class II I-A k using a GGGGSLVPRGSGGGGS (16aa) linker. An interactive view is available in the electronic version of the article. (B) Ribbon representation of TCR HA 1.7 (green)-Hemagglutinin (HA) peptide (yellow)-MHC molecule HLA-DR1 (magenta) complex (PDB code: 1FYT), where the C-terminus of the HA peptide is linked to the N-terminus of the variable β chain of the TCR HA 1.7 using a GGSGGGGG (8aa) linker. The blue dotted line shows the possible positions of the missing amino acids of the linker. An interactive view is available in the electronic version of the article. PRO2206 Figure 4a PRO2206 Figure 4b

Phosphoprotein-nucleocapsid protein complex

Within the virion of the paramyxovirus family of viruses, the nucleocapsid protein (N) packs the genomic RNA into a helical protein-RNA complex known as a nucleocapsid. It is used as a template for the transcription of mRNAs by a viral RNA polymerase, which consists of two components: the large protein and the phosphoprotein (P). While the large protein is involved in the catalytic activities of the polymerase, the phosphoprotein is involved in binding the polymerase to the nucleocapsid through its C-terminal region (459–507). The binding site for the phosphoprotein has been mapped to the C-terminus (486–505) of the nucleocapsid protein. 84 Kinetic studies showed that this is a fast-associating reaction, with a weak binding affinity. Hence, to stabilize this complex, the C-terminus of protein P was linked using a Gly- and Ser-rich linker (GSGSGSGS) to the N-terminus of protein N, and the linked complex was crystallized. This structure showed anti-parallel packing of the helix from the N protein [Fig. 5(A)]. However, the angle between the N protein helix and the final helix of the P protein suggests that it could be an intermolecular interaction between P and N proteins instead of intramolecular interaction. 14

Structural details of transient protein–protein interactions using Gly-rich linkers. (A) Ribbon representation of the crystal structure of phosphoprotein (P457–507) of paramyxoviral polymerase (green)–nucleocapsid protein (N486–505) (magenta), where the C-terminus of P457–507 is linked using a GSGSGSGS (8aa) linker to the N-terminus of the nucleocapsid protein (N486–505) (PDB code: 1T6O). The interacting peptide (magenta) was from the adjacent symmetry-related molecule (cyan). (B) Ribbon representation of the N1 domain of g3p (magenta)—D3 domain of TolA (green) complex, where the C-terminus of g3p is linked using a GGGSEGGGSEGGGSEGGG (18aa) linker to the N-terminus of TolA (PDB code: 1TOL). (C) Ribbon representation of Anti-silencing function 1 (Asf1 green)–α3 helix of Histone H3 (magenta) complex (PDB code: 2IDC). The linker amino acids (AAGAATAA (8aa)) were not present in the pdb except for first two Ala residues. The predicted linker position is shown as a blue dotted line in all panels.

Ribosomes-SRP-FtsY Complex

Escherichia coli signal recognition particle (SRP) consists of a protein (Ffh) and 4.5S RNA. Ffh and FtsY (receptor of SRP) consist of an NG domain, a GTPase G domain, and an N domain. An association between SRP and FtsY via the NG domain is required for the delivery of ribosome-nascent chain complexes to the membrane, and a reciprocal GTPase activation coordinates the transfer of the signal sequence to the translocon. 59 Associations of SRP and FtsY involve a series of GTP-independent and -dependent steps, and form a relatively unstable complex that is visualized by cryo-EM. However, the interaction between Ffh and FtsY is stabilized by fusion of the C-terminus of FtsY to the N-terminus of Ffh via a 31-residue Gly- and Ser- rich linker, yielding a single-chain construct (scSRP). 59 The single chain scSRP behaves similar to an unlinked SRP and FtsY, and binds ribosomes efficiently, without altering the GTPase activity. The cryo-EM structure of scSRP bound to the ribosome was thus described. 59

G3p-TolA complex

Ff phages are used as a selection tool by displaying proteins and peptides on the phage surface by genetic fusion with minor coat gene 3 proteins (g3p). g3p consists of three domains (N1, N2, and CT) connected to each other by natural, flexible Gly-rich linkers. Phage infection of E. coli cell progresses by the interaction between the N2 domain and the tip of an F pilus, with the pilus retracted by an unknown mechanism. 85 At this juncture, the N1 domain interacts with the C-terminal (D3) domain of the integral membrane protein TolA, an interaction which is not clearly understood, and leads to entry of the phage genome into the bacterial cytoplasm. To further understand the mechanism of host cell infection by phages, the interaction between the N1 domain of g3p and the D3 domain of TolA was required. 6 However, the affinity between these two domains is weak. 86 Hence, a Gly-rich linker ((G3SE)3G3) was generated to fuse the C-terminus of the N1 domain of g3p and the N-terminus of the D3 domain of TolA. A long flexible linker was naturally present between the N1 and N2 domains of g3p, and hence the use of a long linker between the N1 domain of g3p and the D3 domain of TolA did not impose any constraints [Fig. 5(B)].

Histone H3-Asf1 complex

Chromatin structure and organization is important during the various stages of the cell cycle. The initial steps of chromatin organization involve the deposition of a (H3/H4)2 heterotetramer onto the DNA. Asf1 (anti-silencing function 1) is one of the several histone chaperones involved in this deposition. NMR and two-hybrid experiments have shown that the C-terminus of the α3 helix of yeast histone H3 interacts with Asf1, 87 , 88 but this interaction is weak. 87 Hence, to obtain a stable complex, the α3 helix of Histone H3 was linked using an alanine-rich linker (AAGAATAA) to Asf1 60 [Fig. 5(C)]. To our knowledge this is the only case where an alanine-rich linker was used.

CaM-calcineurin CBD complex

A similar approach was adopted to study the complex structure of calmodulin (CaM) and the calmodulin-binding domain (CBD) of calcineurin. Calcineurin is a CaM-dependent serine/threonine protein phosphatase involved in various signaling pathways. 89 The CBD of calcineurin is largely disordered in the absence of CaM and attains a secondary structure upon its interaction with CaM. 90 To facilitate the co-crystallization of the CaM-CBD peptide complex, the C-terminus of CaM was linked to the N-terminus of the CBD peptide using a five-amino acid Gly linker (GGGGG). 61 Later, the same group determined the crystal structure of CaM-CBD peptide by co-crystallization in the absence of a linker (PDB: 2R28) and found that the structures of CaM-CBD peptide with and without the linker were same (Fig. 6) this result proved that the presence of the flexible Gly-rich linker did not restrain the interactions between CaM and CBD peptide. 91

Structural similarities between linked and unlinked complexes of Calmodulin and the Calcineurin calmodulin binding domain (CBD). (A) Ribbon representation of Calmodulin (green)–CBD of Calcineurin (magenta) complex, where the C-terminal of Calmodulin is linked using a GGGGG (5aa) linker to the N-terminus of the Calcineurin CBD (PDB code: 2F2P). The predicted linker position is shown as a blue dotted line. (B) Ribbon representation of Calmodulin (green)–CBD of Calcineurin (cyan) complex, generated by co-crystallization (PDB code: 2R28). Both structures were found to be similar. Ca 2+ ions are shown as orange spheres.

Linkers to retain dimerization

Functional dimers are generated by fusing two monomers via a Gly-rich linker, and they are often involved in various enzymatic activities. The HIV-1 protease (HIV-PR) and other retroviral proteases are dimeric molecules that consist of two identical subunits. Crystal structures of HIV-PR have shown that the dimer interface of the protease contains the amino- and carboxyl-terminal strands of each monomeric subunit. 92 This tethered dimer provides a means to determine the effect of changes to one of the two subunits on the activity of HIV-PR. 15 Thus, a covalently linked dimer of HIV-PR was constructed with a 5-amino acid (GGSSG) linker that connected the C-terminus of one monomer to the N-terminus of the other. This covalently linked dimer showed enzymatic properties very similar to those of the natural dimer and was found to be more stable than the natural HIV-PR dimer at pH 7.0, where the natural dimer dissociates. Figure 7(A) shows an example of covalently linked HIV-1 PR dimer. Later structures using various mutants of the HIV-PR tethered dimer and the structures of HIV-PR tethered dimer in the presence of substrate peptides were determined as shown in Table I. 62-70

Crystal structure of covalently linked dimers. (A) Ribbon representation of covalently linked HIV-1 protease (PR) dimer (PDB code: 1HPX), where the C-terminus of one monomer (magenta) is linked to the N-terminus of another (green) using a GGSSG (5aa) linker (shown as a blue dotted line) in the presence of the protease inhibitor compound, KNI-272 (represented as light blue sticks). (B) Ribbon representation of covalently linked transthyretin dimer (PDB code: 1QWH), where the C-terminus of one monomer (magenta) is linked to the N-terminus of another (green) using a GSGGGTGGGSG (11aa) linker. The missing amino acids of the linker are shown as a blue dotted line.

Similarly, transthyretin (TTR), a carrier of the thyroid hormone, thyroxine, and the holo-retinol binding protein, is known to form a tetramer, with two subunits each consisting of a dimer. TTR amyloid diseases are caused due to misfolding, aggregation and deposition of wild-type TTR in cardiac tissue and other destabilizing mutations that make TTR deposition prone in a variety of tissues. The rate-limiting dissociation of the tetramer into its two subunits is necessary for amyloid fibril formation. 93 Experiments were conducted to determine the effect of tethering these closely interacting subunits on the kinetic stabilization of the tetramer. A tethered construct (TTR-GSGGGTGGGSG-TTR)2 was generated to stabilize the tetramer [Fig. 7(B)]. The linked complex was highly stable and also structurally and functionally similar to wild-type TTR. Besides, it was reported that urea was unable to denature the tethered dimer whereas guanidine hydrochloride treatment was able to denature the tethered dimer the authors suggest that tethered complex is kinetically, rather than thermodynamically, stable. 71 This study also showed that tethering two subunits can dramatically reduce tetramer dissociation.

Linker to fuse domains within a protein

In the mammalian nervous system, fast synaptic transmission between nerve cells is carried out mainly by ionotropic glutamate receptors (iGluRs), a class of transmembrane proteins that form glutamate-gated ion channels. They are composed of distinct channel-forming and ligand binding domains, where many synthetic agonists and antagonists are known to bind. 72-74 The S1 and S2 domains form the core ligand binding region of these proteins. Although S1 and S2 form a ligand binding core, they are separated by three membrane spanning segments. Hence, to generate a ligand binding core, S1 and S2 were covalently linked (Fig. 8). For structural studies of the ligand binding domain, a Gly-Thr dipeptide linker was sufficient to covalently link the S1 and S2 domains of the ligand binding core of the glutamate receptors, and mimic its binding affinity for various agonists. 94

Structural details of the ligand binding core of Glutamate receptors (GluRs). (A) Cartoon representation of the domain structure of glutamate receptor ion channels (GluRs). The S1 and S2 domains are linked using a GT peptide to generate a ligand binding core for structural studies. (B) Ribbon representation of the ligand binding core of GluR2, where the C-terminal of S1 (green) is linked to S2 (magenta) using a GT di peptide (dark blue) in complex with glutamic acid (Red) (PDB code: 1FTJ). Zn 2+ ion is shown as an orange sphere.

Linker selection criteria

The selection of a linker sequence and length is dependent on the construction of functional chimeric proteins, and therefore, the optimal linker length will vary on a case by case basis. De novo linker design will be successful if the site of interaction between the two proteins is approximately known. More often, the existing knowledge of the known homologous complex structures is used to design linkers of an appropriate length that will mimic the natural interaction. The actual distance between the site of the interaction and the nearby N- or C-terminus to which the binding partner can be fused will provide clues for the de novo design of a linker or an appropriate length. Often, binding regions on the surface of proteins can be mapped using various biophysical and/or mutational analyses 95 and, based on these details, linker length and composition can be designed. In some cases, linking two proteins with a linker that is hypothesized to promote an intramolecular interaction of a chimeric protein may not be sufficient. Instead, it can form an intermolecular interaction, similar to the case of the phosphoprotein (P459–507) and the nucleocapsid protein (N486–505). 84 Similar binding was observed in our recent studies with CaM and the Neurogranin IQ motif peptide, where the binding partners are linked. We noticed that the peptide and protein from the adjacent molecules were interacting with each other (unpublished data). Our results and the results of others clearly show that if the linker length is not optimal, the linked partner will engage with an adjacent molecule to retain its specificity and natural interactions (intermolecular interaction). Thus, the linkers should be in an optimum length to promote either intramolecular or intermolecular interactions.

In general, the minimum and maximum lengths of linkers used in the studies reviewed here were between 2 and 31 amino acids (Table I), with a common linker lengths of 5 to 11 amino acids for structural studies. The length of the linker mainly depends on the system under study and a preunderstanding of the binding region may help to predict the approximate length of the linker. Apart from length of the linker, the composition should also be optimized. Not all amino acids make a perfect choice for linkers, since a certain degree of flexibility and hydrophilicity of the linker are important to retain the functions of the individual domains and allow the fused proteins to interact with each other. 4 Gly has low preference to form an α-helix thus, the lack of a sidechain maximizes the freedom of the backbone conformation. 96 Furthermore, Ser and Thr are polar residues that prefer to interact with the solvent than with the fused proteins. 4 Thus, polypeptides rich in Gly, Ser, and Thr offer special advantages: (i) rotational freedom of the polypeptide backbone, so that the adjacent domains are free to move relative to one another, 44 (ii) enhanced solubility, 26 and (iii) resistance to proteolysis. 97 The backbone dihedral angles of residues in linkers are highly variable that is, in Ramachandran plots, they occupy a significantly larger area than either the α-helices or β-sheets.

Our literature analyses show that various compositions of linkers have been used for structural studies (Table I). In the case of MHC Class II-peptide complex, two different types of linkers (with and without thrombin cleavage site) were used, while in the case of MHC-peptide-TCR ternary complex, peptide linking was achieved through two different methods (peptide linked to either MHC or TCR molecule) (Table I). In other structural studies, linkers with almost equal composition of Gly and Ser were used, such as for the phosphoprotein-nucleocapsid protein complex, the ribosomes-SRP-FtsY complex and the Fat domain of focal adhesion kinase complexes. By comparison, for the linked complexes of the LIM domain, PXR-LBD, peptide bound to TCR-MHC, g3p-TolA, HIV-1 PR, and TTR covalently dimers, various lengths of Gly- and Ser-rich linkers were employed, for which over 60% of the linker amino acids were Gly. For structural studies of CaM-CBD of calcineurin complex, the study integrated a 5-amino acids linker with all Gly residues. Hence, a suitable linker might comprise a combination of small side chained amino acids, such as Gly and Ser, with an overall higher percentage of Gly.

Apart from designing linkers (length and amino acid composition), optimizing conditions to retain the interactions between the linked partners is important. In general, the flexible Gly-rich linkers do not alter the properties of the proteins to which they are bound, and permit the natural interaction to be retained. Optimized conditions for the interaction between the linked partners should be the same as that without the linker. This should be confirmed using unlinked interacting partners in various in vitro methods, such as immunoprecipitation or ITC/SPR experiments.

D7. Redox Chemistry and Protein Folding - Biology

The production of recombinant, disulfide-containing proteins often requires oxidative folding in vitro. Here, we show that diselenides, such as selenoglutathione, catalyze oxidative protein folding by O2. Substantially lower concentrations of a redox buffer composed of selenoglutathione and the thiol form of glutathione can consequently be used to achieve the same rate and yield of folding as a standard glutathione redox buffer. Further, the low pKa of selenols extends the pH range for folding by selenoglutathione to acidic conditions, where glutathione is inactive. Harnessing the catalytic power of diselenides may thus pave the way for more efficient oxidative protein folding.

Virus-like Particles as Quantitative Probes of Membrane Protein Interactions
  • Sharon Willis ,
  • Candice Davidoff ,
  • Justin Schilling ,
  • Antony Wanless ,
  • Benjamin J. Doranz , and
  • Joseph Rucker*

We demonstrate that virus-like particles carrying conformationally complex membrane proteins (“lipoparticles”) can be used as soluble probes of membrane protein interactions. To demonstrate the utility of this approach, we use lipoparticles to rapidly differentiate the relative kinetics of membrane protein interactions using optical biosensor technology. The technique is applied to diverse membrane proteins, including G protein-coupled receptors, and used to rank the relative kinetics of nearly all the commercially available monoclonal antibodies against chemokine receptor CCR5. These particles serve as versatile probes for screening crude and purified antibody preparations for receptor specificity, epitope reactivity, and relative binding kinetics.

Current Topics
The Glycosylphosphatidylinositol Anchor: A Complex Membrane-Anchoring Structure for Proteins

Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified protein in the outer leaflet of the cell membrane. The GPI anchor is a complex structure comprising a phosphoethanolamine linker, glycan core, and phospholipid tail. GPI-anchored proteins are structurally and functionally diverse and play vital roles in numerous biological processes. While several GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. This review discusses the structural diversity of the GPI anchor and its putative cellular functions, including involvement in lipid raft partitioning, signal transduction, targeting to the apical membrane, and prion disease pathogenesis. We specifically highlight studies in which chemically synthesized GPI anchors and analogues have been employed to study the roles of this unique posttranslational modification.

Structural and Functional Diversities between Members of the Human HSPB, HSPH, HSPA, and DNAJ Chaperone Families
  • Michel J. Vos ,
  • Jurre Hageman ,
  • Serena Carra , and
  • Harm H. Kampinga*

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins required to deal with proteotoxic stresses. Besides being stress-protective and possible targets for delaying progression of protein folding diseases, mutations in chaperones also have been shown to cause disease (chaperonopathies). The mechanism of action of the “classical”, stress-inducible HSPs in serving as molecular chaperones preventing the irreversible aggregation of stress-unfolded or disease-related misfolded proteins is beginning to emerge. However, the human genome encodes several members for each of the various HSP families that are not stress-related but contain conserved domains. Here, we have reviewed the existing literature on the various members of the human HSPB (HSP27), HSPH (HSP110), HSPA (HSP70), and DNAJ (HSP40) families. Apart from structural and functional homologies, several diversities between members and families can be found that not only point to differences in client specificity but also seem to serve differential client handling and processing. How substrate specificity and client processing is determined is far from being understood.

Accelerated Publications
Light Activation of the LOV Protein Vivid Generates a Rapidly Exchanging Dimer

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap−PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV−LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Quantitative and Real-Time Detection of Secretion of Chemical Messengers from Individual Platelets

Carbon-fiber microelectrochemical methods were utilized in this study to measure individual exocytotic events of secretion of serotonin and histamine from washed rabbit platelets. The quantal release of serotonin was quantitatively characterized with a δ-granule serotonin concentration of 0.6 M and secretion time course of 7 ms. Additionally, extracellular osmolarity influences quantal size, causing quantal size increases under hypotonic conditions, presumably due to the influx of cytosolic serotonin into the halo region of the δ-granules.

Diverse Regulation of SNF2h Chromatin Remodeling by Noncatalytic Subunits
  • Xi He ,
  • Hua-Ying Fan ,
  • Joseph D. Garlick , and
  • Robert E. Kingston*

SNF2h-based ATP-dependent chromatin remodeling complexes diverge in composition, nuclear localization, and biological function. Such differences have led to the hypothesis that SNF2h complexes differ mechanistically. One proposal is that the complexes have different functional interactions with the naked DNA adjacent to the nucleosome. We have used a series of templates with defined nucleosomal position and differing amounts and placement of adjacent DNA to compare the relative activities of SNF2h and SNF2h complexes. The complexes hACF, CHRAC, WICH, and RSF all displayed differences in functional interactions with these templates, which we attribute to the differences in the noncatalytic subunit. We suggest that the ability to sense adjacent DNA is a general property of the binding partners of SNF2h and that each partner provides distinct regulation that contributes to distinct cellular function.

Catalytic Diversity of Extended Hammerhead Ribozymes

Chimeras of the well-characterized minimal hammerhead 16 and nine extended hammerheads derived from natural viroids and satellite RNAs were constructed with the goal of assessing whether their very different peripheral tertiary interactions modulate their catalytic properties. For each chimera, three different assays were used to determine the rate of cleavage and the fraction of full-length hammerhead at equilibrium and thereby deduce the elemental cleavage (k2) and ligation (k−2) rate constants. The nine chimeras were all more active than minimal hammerheads and exhibited a very broad range of catalytic properties, with values of k2 varying by 750-fold and k−2 by 100-fold. At least two of the hammerheads exhibited an altered dependence of kobs on magnesium concentration. Since much less catalytic diversity is observed among minimal hammerheads that lack the tertiary interactions, a possible role for the different tertiary interaction is to modulate the hammerhead cleavage properties in viroids. For example, differing hammerhead cleavage and ligation rates could affect the steady state concentrations of linear, circular, and polymeric genomes in infected cells.

Local RNA Conformational Dynamics Revealed by 2-Aminopurine Solvent Accessibility
  • Jeff D. Ballin ,
  • James P. Prevas ,
  • Shashank Bharill ,
  • Ignacy Gryczynski ,
  • Zygmunt Gryczynski , and
  • Gerald M. Wilson*

Acrylamide quenching is widely used to monitor the solvent exposure of fluorescent probes in vitro. Here, we tested the utility of this technique to discriminate local RNA secondary structures using the fluorescent adenine analogue 2-aminopurine (2-AP). Under native conditions, the solvent accessibilities of most 2-AP-labeled RNA substrates were poorly resolved by classical single-population models rather, a two-state quencher accessibility algorithm was required to model acrylamide-dependent changes in 2-AP fluorescence in structured RNA contexts. Comparing 2-AP quenching parameters between structured and unstructured RNA substrates permitted the effects of local RNA structure on 2-AP solvent exposure to be distinguished from nearest neighbor effects or environmental influences on intrinsic 2-AP photophysics. Using this strategy, the fractional accessibility of 2-AP for acrylamide (fa) was found to be highly sensitive to local RNA structure. Base-paired 2-AP exhibited relatively poor accessibility, consistent with extensive shielding by adjacent bases. 2-AP in a single-base bulge was uniformly accessible to solvent, whereas the fractional accessibility of 2-AP in a hexanucleotide loop was indistinguishable from that of an unstructured RNA. However, these studies also provided evidence that the fa parameter reflects local conformational dynamics in base-paired RNA. Enhanced base pair dynamics at elevated temperatures were accompanied by increased fa values, while restricting local RNA breathing by adding a C-G base pair clamp or positioning 2-AP within extended RNA duplexes significantly decreased this parameter. Together, these studies show that 2-AP quenching studies can reveal local RNA structural and dynamic features beyond those that can be measured by conventional spectroscopic approaches.

Acetylation of H4 Suppresses the Repressive Effects of the N-Termini of Histones H3/H4 and Facilitates the Formation of Positively Coiled DNA

We have studied the role of the N-termini of histones H3/H4 in the regulation of the conformational changes that occur in H3/H4 during their deposition on DNA by NAP1 (nucleosome assembly protein 1). Removal of the N-termini extensively increased the right-handed conformation of H3/H4 as assayed by the increased levels of positive coils that were formed on DNA. The osmolytes, TMAO, betaine, sarcosine, alanine, glycine, and proline to varying degrees, facilitated the formation of positive coils. The denaturant, urea (0.6 M), blocked the osmolyte effects, causing a preference of H3/H4 to form negative coils (the left-handed conformation). Acetylated H3/H4 also formed high levels of positive coils, and it is proposed that both the osmolytes and acetylation promote the formation of an α-helix in the N-termini. This structural change may ultimately explain a unique feature of transcription through nucleosomes, i.e., that H2A/H2B tends to be more mobile than H3/H4. By using combinations of H3 and H4 that were either acetylated or the N-termini removed, it was also determined that the N-terminus of H4 is primarily responsible for repressing the formation of positive coils. Additional gradient analyses indicate that NAP1 establishes an equilibrium with the H3/H4−DNA complexes. This equilibrium facilitates a histone saturation of the DNA, a unique state that promotes the right-handed conformation. NAP1 persists in the binding of the complexes through interaction with the N-terminus of H3, which may be a mechanism for subsequent remodeling of the nucleosome during transcription and replication.

The NMR Structure and Dynamics of the Two-Domain Tick Carboxypeptidase Inhibitor Reveal Flexibility in Its Free Form and Stiffness upon Binding to Human Carboxypeptidase B
  • David Pantoja-Uceda ,
  • Joan L. Arolas ,
  • Pascal García ,
  • Eva López-Hernández ,
  • Daniel Padró ,
  • Francesc X. Aviles* , and
  • Francisco J. Blanco*

The structure of the tick carboxypeptidase inhibitor (TCI) and its backbone dynamics, free and in complex with human carboxypeptidase B, have been determined by NMR spectroscopy. Although free TCI has the same overall fold as that observed in the crystal structures of its complexes with metallocarboxypeptidase types A and B, there are structural differences at the linker between the two domains. The linker residues have greater flexibility than the globular domains, and the C-terminal residues are highly flexible in free TCI. Upon formation of a complex with carboxypeptidase B, TCI becomes more rigid, especially at the level of the linker and at the C-terminus, which is inserted into the active site groove of the carboxypeptidase. Solvent exchange rates of the backbone amide protons also show a strong reduction of the local TCI dynamics and a stabilization of its structure upon complex formation. The findings are consistent with a recognition mechanism that primarily involves the C-terminal domain, which adjusts its conformation and that of the linker, thus facilitating complex stabilization by further interactions between the N-terminal domain and an exosite of the carboxypeptidase. This adaptability enables TCI to tune its global conformation for proper interaction with distinct types of carboxypeptidases by a mechanism of induced fit. Our results provide new information about the structure−function relationship and stability of a molecule with potential biomedical applications in thrombolytic therapy. Furthermore, the plasticity of TCI makes it an ideal scaffold for developing stronger and/or more specific inhibitors directed toward modulating the activity of metallocarboxypeptidases.

Microscopic Reversibility of Protein Folding in Molecular Dynamics Simulations of the Engrailed Homeodomain

The principle of microscopic reversibility states that at equilibrium the number of molecules entering a state by a given path must equal those exiting the state via the same path under identical conditions or, in structural terms, that the conformations along the two pathways are the same. There has been some indirect evidence indicating that protein folding is such a process, but there have been few conclusive findings. In this study, we performed molecular dynamics simulations of an ultrafast unfolding and folding protein at its melting temperature to observe, on an atom-by-atom basis, the pathways the protein followed as it unfolded and folded within a continuous trajectory. In a total of 0.67 µs of simulation in water, we found six transient denaturing events near the melting temperature (323 and 330 K) and an additional refolding event following a previously identified unfolding event at a high temperature (373 K). In each case, unfolding and refolding transition state ensembles were identified, and they agreed well with experiment on the basis of a comparison of S and Φ values. On the basis of several structural properties, these 13 transition state ensembles agreed very well with each other and with four previously identified transition states from high-temperature denaturing simulations. Thus, not only were the unfolding and refolding transition states part of the same ensemble, but in five of the seven cases, the pathway the protein took as it unfolded was nearly identical to the subsequent refolding pathway. These events provide compelling evidence that protein folding is a microscopically reversible process. In the other two cases, the folding and unfolding transition states were remarkably similar to each other but the paths deviated.

Cholesterol Is Found To Reside in the Center of a Polyunsaturated Lipid Membrane

Previously, we reported neutron diffraction studies on the depth of cholesterol in phosphatidylcholine (PC) bilayers with varying amounts of acyl chain unsaturation [Harroun, T. A., et al. (2006) Biochemistry 45, 1227−1233]. The center of mass of the 2,2,3,4,4,6-D6 deuterated sites on the sterol label was found to reside 16 Å from the middle of the bilayer in 1-palmitoyl-2-oleoylphosphatidylcholine (16:0-18:1PC), 1,2-dioleoylphosphatidylcholine (18:1-18:1PC), and 1-stearoyl-2-arachidonylphosphatidylcholine (18:0-20:4PC). This location places cholesterol’s hydroxyl group close to the membrane surface, indicative of the molecule in its commonly understood “upright” orientation. However, for dipolyunsaturated 20:4-20:4PC membranes the label, thus the hydroxyl group, was found sequestered in the center of the bilayer. We attributed the change in location to the high level of disorder of polyunsaturated fatty acids (PUFA) that is incompatible with proximity to the rigid steroid moiety in its usual upright orientation. From that study, the unresolved question was whether the molecule was inverted or lying flat with respect to the membrane plane, in the middle of the bilayer. We have followed up those results with additional neutron experiments employing [25,26,26,26,27,27-D7]cholesterol, a deuterated analogue labeled in the tail. These diffraction measurements unequivocally show cholesterol lies flat in the middle of 20:4-20:4PC bilayers.

Characterization of a Streptococcal Cholesterol-Dependent Cytolysin with a Lewis y and b Specific Lectin Domain
  • Stephen Farrand ,
  • Eileen Hotze ,
  • Paul Friese ,
  • Susan K. Hollingshead ,
  • David F. Smith ,
  • Richard D. Cummings ,
  • George L. Dale , and
  • Rodney K. Tweten*

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that often exhibit distinct structural changes that modify their pore-forming activity. A soluble platelet aggregation factor from Streptococcus mitis (Sm-hPAF) was characterized and shown to be a functional CDC with an amino-terminal fucose-binding lectin domain. Sm-hPAF, or lectinolysin (LLY) as renamed herein, is most closely related to CDCs from Streptococcus intermedius (ILY) and Streptococcus pneumoniae (pneumolysin or PLY). The LLY gene was identified in strains of S. mitis, S. pneumoniae, and Streptococcus pseudopneumoniae. LLY induces pore-dependent changes in the light scattering properties of the platelets that mimic those induced by platelet aggregation but does not induce platelet aggregation. LLY monomers form the typical large homooligomeric membrane pore complex observed for the CDCs. The pore-forming activity of LLY on platelets is modulated by the amino-terminal lectin domain, a structure that is not present in other CDCs. Glycan microarray analysis showed the lectin domain is specific for difucosylated glycans within Lewis b (Leb) and Lewis y (Ley) antigens. The glycan-binding site is occluded in the soluble monomer of LLY but is apparently exposed after cell binding, since it significantly increases LLY pore-forming activity in a glycan-dependent manner. Hence, LLY represents a new class of CDC whose pore-forming mechanism is modulated by a glycan-binding domain.

Monooxygenase Activity of Octopus vulgaris Hemocyanin
  • Kenji Suzuki ,
  • Chizu Shimokawa ,
  • Chiyuki Morioka , and
  • Shinobu Itoh*

Octopus vulgaris hemocyanin (Ov-Hc) and one of its minimal functional units (Ov-g) have been purified, and their spectroscopic features and monooxygenase (phenolase) activity have been examined in detail. The oxy forms of both Ov-Hc and Ov-g are stable in 0.5 M borate buffer (pH 9.0) even in the presence of a high concentration of urea at 25 °C ∼90 and ∼75% of the (μ-η2:η2-peroxo)dicopper(II) species of Ov-Hc and Ov-g, respectively, remained unchanged after argon (Ar) gas flushing of the sample solutions for 1 h. The catalytic activity of Ov-g in the oxygenation reaction (multiturnover reaction) of 4-methylphenol (p-cresol) to 4-methyl-1,2-dihydroxybenzene (4-methylcatechol) was higher than that of Ov-Hc, and its catalytic activity was further accelerated by the addition of urea. Kinetic deuterium isotope effect analysis and Hammett analysis using a series of phenol derivatives under anaerobic conditions (single-turnover reaction) have indicated that the monooxygenation reaction of phenols to catechols by the peroxo species of oxyhemocyanin proceeds via electrophilic aromatic substitution mechanism as in the case of tyrosinase. The effect of urea on the redox functions of oxyhemocyanin is discussed on the basis of the spectroscopic analysis and reactivity studies.

Thermodynamic Analysis of Denaturant-Induced Unfolding of HodC69S Protein Supports a Three-State Mechanism
  • Kristian Boehm ,
  • Jessica Guddorf ,
  • Alexander Albers ,
  • Tadashi Kamiyama ,
  • Susanne Fetzner , and
  • H.-J. Hinz*

Thermodynamic stability parameters and the equilibrium unfolding mechanism of His6HodC69S, a mutant of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) having a Cys to Ser exchange at position 69 and an N-terminal hexahistidine tag (His6HodC69S), have been derived from isothermal unfolding studies using guanidine hydrochloride (GdnHCl) or urea as denaturants. The conformational changes were monitored by following changes in circular dichroism (CD), fluorescence, and dynamic light scattering (DLS), and the resulting transition curves were analyzed on the basis of a sequential three-state model N = I = D. The structural changes have been correlated to catalytic activity, and the contribution to stability of the disulfide bond between residues C37 and C184 in the native protein has been established. A prominent result of the present study is the finding that, independent of the method used for denaturing the protein, the unfolding mechanism always comprises three states which can be characterized by, within error limits, identical sets of thermodynamic parameters. Apparent deviations from three-state unfolding can be rationalized by the inability of a spectroscopic probe to discriminate clearly between native, intermediate, and unfolded ensembles. This was the case for the CD-monitored urea unfolding curve.

Identification of a Minimal Functional Linker in Human Topoisomerase I by Domain Swapping with Cre Recombinase
  • Rikke From Frøhlich ,
  • Sissel Juul ,
  • Maria Bjerre Nielsen ,
  • Maria Vinther ,
  • Christopher Veigaard ,
  • Marianne Smedegaard Hede , and
  • Félicie Faucon Andersen*

Cellular forms of type IB topoisomerases distinguish themselves from their viral counterparts and the tyrosine recombinases to which they are closely related by having rather extensive N-terminal and linker domains. The functions and necessity of these domains are not yet fully unraveled. In this study we replace 86 amino acids including the linker domain of the cellular type IB topoisomerase, human topoisomerase I, with four, six, or eight amino acids from the corresponding short loop region in Cre recombinase. In vitro characterization of the resulting chimeras, denoted Cropos, reveals that six amino acids from the Cre linker loop constitute the minimal length of a functional linker in human topoisomerase I.

Superior Removal of Hydantoin Lesions Relative to Other Oxidized Bases by the Human DNA Glycosylase hNEIL1
  • Nirmala Krishnamurthy ,
  • Xiaobei Zhao ,
  • Cynthia J. Burrows , and
  • Sheila S. David*

The DNA glycosylase hNEIL1 initiates the base excision repair (BER) of a diverse array of lesions, including ring-opened purines and saturated pyrimidines. Of these, the hydantoin lesions, guanidinohydantoin (Gh) and the two diastereomers of spiroiminodihydantoin (Sp1 and Sp2), have garnered much recent attention due to their unusual structures, high mutagenic potential, and detection in cells. In order to provide insight into the role of repair, the excision efficiency by hNEIL1 of these hydantoin lesions relative to other known substrates was determined. Most notably, quantitative examination of the substrate specificity with hNEIL1 revealed that the hydantoin lesions are excised much more efficiently (>100-fold faster) than the reported standard substrates thymine glycol (Tg) and 5-hydroxycytosine (5-OHC). Importantly, the glycosylase and β,δ-lyase reactions are tightly coupled such that the rate of the lyase activity does not influence the observed substrate specificity. The activity of hNEIL1 is also influenced by the base pair partner of the lesion, with both Gh and Sp removal being more efficient when paired with T, G, or C than when paired with A. Notably, the most efficient removal is observed with the Gh or Sp paired in the unlikely physiological context with T indeed, this may be a consequence of the unstable nature of base pairs with T. However, the facile removal via BER in promutagenic base pairs that are reasonably formed after replication (such as Gh·G) may be a factor that modulates the mutagenic profile of these lesions. In addition, hNEIL1 excises Sp1 faster than Sp2, indicating the enzyme can discriminate between the two diastereomers. This is the first time that a BER glycosylase has been shown to be able to preferentially excise one diastereomer of Sp. This may be a consequence of the architecture of the active site of hNEIL1 and the structural uniqueness of the Sp lesion. These results indicate that the hydantoin lesions are the best substrates identified thus far for hNEIL1 and suggest that repair of these lesions may be a critical function of the hNEIL1 enzyme in vivo.

A Bridging Water Anchors the Tethered 5-(3-Aminopropyl)-2′-deoxyuridine Amine in the DNA Major Groove Proximate to the N+2 C·G Base Pair: Implications for Formation of Interstrand 5′-GNC-3′ Cross-Links by Nitrogen Mustards ,
  • Feng Wang ,
  • Feng Li ,
  • Manjori Ganguly ,
  • Luis A. Marky ,
  • Barry Gold ,
  • Martin Egli , and
  • Michael P. Stone*

Site-specific insertion of 5-(3-aminopropyl)-2′-deoxyuridine (Z3dU) and 7-deaza-dG into the Dickerson−Drew dodecamers 5′-d(C1G2C3G4A5A6T7T8C9Z10C11G12)-3′·5′-d(C13G14C15G16A17A18T19T20C21Z22C23G24)-3′ (named DDDZ10) and 5′-d(C1G2C3G4A5A6T7X8C9Z10C11G12)-3′·5′-d(C13G14C15G16A17A18T19X20C21Z22C23G24)-3′ (named DDD2+Z10) (X = Z3dU Z = 7-deaza-dG) suggests a mechanism underlying the formation of interstrand N+2 DNA cross-links by nitrogen mustards, e.g., melphalan and mechlorethamine. Analysis of the DDD2+Z10 duplex reveals that the tethered cations at base pairs A5·X20 and X8·A17 extend within the major groove in the 3′-direction, toward conserved Mg2+ binding sites located adjacent to N+2 base pairs C3·Z22 and Z10·C15. Bridging waters located between the tethered amines and either Z10 or Z22 O6 stabilize the tethered cations and allow interactions with the N + 2 base pairs without DNA bending. Incorporation of 7-deaza-dG into the DDD2+Z10 duplex weakens but does not eliminate electrostatic interactions between tethered amines and Z10 O6 and Z22 O6. The results suggest a mechanism by which tethered N7-dG aziridinium ions, the active species involved in formation of interstrand 5′-GNC-3′ cross-links by nitrogen mustards, modify the electrostatics of the major groove and position the aziridinium ions proximate to the major groove edge of the N+2 C·G base pair, facilitating interstrand cross-linking.

Solution X-ray Scattering Reveals a Novel Structure of Calmodulin Complexed with a Binding Domain Peptide from the HIV-1 Matrix Protein p17
  • Yoshinobu Izumi* ,
  • Hiroki Watanabe ,
  • Noriko Watanabe ,
  • Aki Aoyama ,
  • Yuji Jinbo , and
  • Nobuhiro Hayashi

The solution structures of complexes between calcium-saturated calmodulin (Ca2+/CaM) and a CaM-binding domain of the HIV-1 matrix protein p17 have been determined by small-angle X-ray scattering with use of synchrotron radiation as an intense and stable X-ray source. We used three synthetic peptides of residues 11−28, 26−47, and 11−47 of p17 to demonstrate the diversity of CaM-binding conformation. Ca2+/CaM complexed with residues 11−28 of p17 adopts a dumbbell-like structure at a molar ratio of 1:2, suggesting that the two peptides bind each lobe of CaM, respectively. Ca2+/CaM complexed with residues 26−47 of p17 at a molar ratio of 1:1 adopts a globular structure similar to the NMR structure of Ca2+/CaM bound to M13, which adopted a compact globular structure. In contrast to these complexes, Ca2+/CaM binds directly with both CaM-binding sites of residues 11−47 of p17 at a molar ratio of 1:1, which induces a novel structure different from known structures previously reported between Ca2+/CaM and peptide. A tertiary structural model of the novel structure was constructed using the biopolymer module of Insight II 2000 on the basis of the scattering data. The two domains of CaM remain essentially unchanged upon complexation. The hinge motions, however, occur in a highly flexible linker of CaM, in which the electrostatic residues 74Arg, 78Asp, and 82Glu interact with N-terminal electrostatic residues of the peptide (residues 12Glu, 15Arg, and 18Lys). The acidic residues in the N-terminal domain of CaM interact with basic residues in a central part of the peptide, thereby enabling the central part to change the conformations, while an acidic residue in the C-terminal domain interacts with two basic residues in the two helical sites of the peptide. The overall structure of the complex adopts an extended structure with the radius of gyration of 20.5 Å and the interdomain distance of 34.2 Å. Thus, the complex is principally stabilized by electrostatic interactions. The hydrophobic patches of Ca2+/CaM are not responsible for the binding with the hydrophobic residues in the peptide, suggesting that CaM plays a role to sequester the myristic acid moiety of p17.

The trans-Golgi Proteins SCLIP and SCG10 Interact with Chromogranin A To Regulate Neuroendocrine Secretion
  • Nitish R. Mahapatra ,
  • Laurent Taupenot* ,
  • Maite Courel ,
  • Sushil K. Mahata* , and
  • Daniel T. O’Connor*

Secretion of proteins and peptides from eukaryotic cells takes place by both constitutive and regulated pathways. Regulated secretion may involve interplay of proteins that are currently unknown. Recent studies suggest an important role of chromogranin A (CHGA) in the regulated secretory pathway in neuroendocrine cells, but the mechanism by which CHGA enters the regulated pathway, or even triggers the formation of the pathway, remains unclear. In this study, we used a transcriptome/proteome-wide approach, to discover binding partners for CHGA, by employing a phage display cDNA library method. Several proteins within or adjacent to the secretory pathway were initially detected as binding partners of recombinant human CHGA. We then focused on the trans-Golgi protein SCLIP (STMN3) and its stathmin paralog SCG10 (STMN2) for functional study. Co-immunoprecipitation experiments confirmed the interaction of each of these two proteins with CHGA in vitro. SCLIP and SCG10 were colocalized to the Golgi apparatus of chromaffin cells in vivo and shared localization with CHGA as it transited the Golgi. Downregulation of either SCLIP or SCG10 by synthetic siRNAs virtually abolished chromaffin cell secretion of a transfected CHGA−EAP chimera (expressing CHGA fused to an enzymatic reporter, and trafficked to the regulated pathway). SCLIP siRNA also decreased the level of secretion of endogenous CHGA and SCG2, as well as transfected human growth hormone, while SCG10 siRNA decreased the level of regulated secretion of endogenous CHGB. Moreover, a dominant negative mutant of SCG10 (Cys22,Cys24→Ala22,Ala24) significantly blocked secretion of the transfected CHGA−EAP chimera. A decrease in the buoyant density of chromaffin granules was observed after downregulation of SCG10 by siRNA, suggesting participation of these stathmins in granule formation or maturation. We conclude that SCLIP and SCG10 interact with CHGA, share partial colocalization in the Golgi apparatus, and may be necessary for typical transmitter storage and release from chromaffin cells.

Molecular Basis for Tetanus Toxin Coreceptor Interactions

Tetanus toxin (TeNT) elicits spastic paralysis through the cleavage of vesicle-associated membrane protein-2 (VAMP-2) in neurons at the interneuronal junction of the central nervous system. While TeNT retrograde traffics from peripheral nerve endings to the interneuronal junction, there is limited understanding of the neuronal receptors utilized by tetanus toxin for the initial entry into nerve cells. Earlier studies implicated a coreceptor for tetanus toxin entry into neurons: a ganglioside binding pocket and a sialic acid binding pocket and that GT1b bound to each pocket. In this study, a solid phase assay characterized the ganglioside binding specificity and functional properties of both carbohydrate binding pockets of TeNT. The ganglioside binding pocket recognized the ganglioside sugar backbone, Gal-GalNAc, independent of sialic acid-(5) and sialic acid-(7) and GM1a was an optimal substrate for this pocket, while the sialic acid binding pocket recognized sialic acid-(5) and sialic acid-(7) with “b”series of gangliosides preferred relative to “a” series gangliosides. The high-affinity binding of gangliosides to TeNT HCR required functional ganglioside and sialic acid binding pockets, supporting synergistic binding to coreceptors. This analysis provides a model for how tetanus toxin utilizes coreceptors for high-affinity binding to neurons.

Insights into the Mechanism of Oxidative Deamination Catalyzed by DOPA Decarboxylase
  • Mariarita Bertoldi* ,
  • Barbara Cellini ,
  • Riccardo Montioli , and
  • Carla Borri Voltattorni

The unusual oxygen-consuming oxidative deamination reaction catalyzed by the pyridoxal 5′-phosphate (PLP) enzyme DOPA decarboxylase (DDC) was here investigated. Either wild-type or Y332F DDC variant is able to perform such oxidation toward aromatic amines or aromatic l-amino acids, respectively, without the aid of any cofactor related to oxygen chemistry. Oxidative deamination produces, in equivalent amounts, a carbonyl compound and ammonia, accompanied by dioxygen consumption in a 1:2 molar ratio with respect to the products. Kinetic studies either in the pre-steady or in the steady state, together with HPLC analyses of reaction mixtures under varying experimental conditions, revealed that a ketimine accumulates during the linear phase of product formation. This species is reactive since it is converted back to PLP when the substrate is consumed. Rapid-mixing chemical quench studies provide evidence that the ketimine is indeed an intermediate formed during the first catalytic cycle. Moreover, superoxide anion and hydrogen peroxide are both generated during the catalytic cycles. On this basis, a mechanism of oxidative deamination consistent with the present data is proposed. Furthermore, the catalytic properties of the T246A DDC mutant together with those previously obtained with H192Q mutant allow us to propose that the Thr246-His192 dyad could act as a general base in promoting the first step of the oxidative deamination of aromatic amines.

Thermodynamics of the Dimer−Decamer Transition of Reduced Human and Plant 2-Cys Peroxiredoxin
  • Sergio Barranco-Medina ,
  • Sergej Kakorin ,
  • Juan José Lázaro , and
  • Karl-Josef Dietz*

Isothermal titration calorimetry (ITC) is a powerful technique for investigating self-association processes of protein complexes and was expected to reveal quantitative data on peroxiredoxin oligomerization by directly measuring the thermodynamic parameters of dimer−dimer interaction. Recombinant classical 2-cysteine peroxoredoxins from Homo sapiens, Arabidopsis thaliana, and Pisum sativum as well as a carboxy-terminally truncated variant were subjected to ITC analysis by stepwise injection into the reaction vessel under various redox conditions. The direct measurement of the decamer−dimer equilibrium of reduced peroxiredoxin revealed a critical concentration in the very low micromolar range. The data suggest a cooperative assembly above this critical transition concentration where a nucleus facilitates assembly. The rather abrupt transition indicates that assembly processes do not occur below the critical transition concentration while oligomerization is efficiently triggered above it. The magnitude of the measured enthalpy confirmed the endothermic nature of the peroxiredoxin oligomerization. Heterocomplexes between peroxiredoxin polypeptides from different species were not formed. We conclude that a functional constraint conserved the dimer−decamer transition with highly similar critical transition concentrations despite emerging sequence variation during evolution.

Probing the Steroid Binding Domain-like I (SBDLI) of the Sigma-1 Receptor Binding Site Using N-Substituted Photoaffinity Labels
  • Dominique Fontanilla ,
  • Abdol R. Hajipour ,
  • Arindam Pal ,
  • Uyen B. Chu ,
  • Marty Arbabian , and
  • Arnold E. Ruoho*

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[125I]iodo-4-azidococaine ([125I]IACoc) binds to the sigma-1 receptor with 2−3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393−1397]. Using this photoprobe, we demonstrated the insertion site for [125I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532−3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176−194) [Pal, A. (2007) Mol. Pharmacol. 72, 921−933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1-N-(2-3-[125I]iodophenyl)propane ([125I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91−109) [Pal, A. (2007) Mol. Pharmacol. 72, 921−933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative “nitrogen interacting region” of sigma-1 receptor ligands. These two novel photoprobes are (−)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[125I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([125I]-N-IACoc) and N-propyl-N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([125I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283−292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [125I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [125I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.

Role of Conserved Aspartates in the ArsA ATPase
  • Hiranmoy Bhattacharjee* ,
  • Ranginee Choudhury , and
  • Barry P. Rosen

The ArsA ATPase is the catalytic subunit of the arsenite-translocating ArsAB pump that is responsible for resistance to arsenicals and antimonials in Escherichia coli. ATPase activity is activated by either arsenite or antimonite. ArsA is composed of two homologous halves A1 and A2, each containing a nucleotide binding domain, and a single metalloid binding or activation domain is located at the interface of the two halves of the protein. The metalloid binding domain is connected to the two nucleotide binding domains through two DTAPTGH sequences, one in A1 and the other in A2. The DTAPTGH sequences are proposed to be involved in information communication between the metal and catalytic sites. The roles of Asp142 in A1 D142TAPTGH sequence, and Asp447 in A2 D447TAPTGH sequence was investigated after altering the aspartates individually to alanine, asparagine, and glutamate by site-directed mutagenesis. Asp142 mutants were sensitive to As(III) to varying degrees, whereas the Asp447 mutants showed the same resistance phenotype as the wild type. Each altered protein exhibited varying levels of both basal and metalloid-stimulated activity, indicating that neither Asp142 nor Asp447 is essential for catalysis. Biochemical characterization of the altered proteins imply that Asp142 is involved in Mg2+ binding and also plays a role in signal transduction between the catalytic and activation domains. In contrast, Asp447 is not nearly as critical for Mg2+ binding as Asp142 but appears to be in communication between the metal and catalytic sites. Taken together, the results indicate that Asp142 and Asp447, located on the A1 and A2 halves of the protein, have different roles in ArsA catalysis, consistent with our proposal that these two halves are functionally nonequivalent.

The Regulatory β Subunit of Phosphorylase Kinase Interacts with Glyceraldehyde-3-phosphate Dehydrogenase
  • Igor G. Boulatnikov ,
  • Owen W. Nadeau ,
  • Patrick J. Daniels ,
  • Jessica M. Sage ,
  • Marina D. Jeyasingham ,
  • Maria T. Villar ,
  • Antonio Artigues , and
  • Gerald M. Carlson*

Skeletal muscle phosphorylase kinase (PhK) is an (αβγδ)4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GPb) in a Ca2+-dependent reaction that couples muscle contraction with glycogen breakdown. GPb is PhK’s only known in vivo substrate however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [γ-32P]ATP with or without Ca2+ and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca2+-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC50 ∼ 0.45 μM) PhK’s conversion of GPb. When a short synthetic peptide substrate was substituted for GPb, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the γ subunit. To test this notion, the PhK−GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory β subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the β subunit, specifically an αγδ subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GPb. Moreover, GAPDH had no effect on the conversion of GPb by the αγδ subcomplex. The interactions described herein between the β subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.

Exploring the Energy Landscape of Antibody−Antigen Complexes: Protein Dynamics, Flexibility, and Molecular Recognition
  • Megan C. Thielges ,
  • Jörg Zimmermann ,
  • Wayne Yu ,
  • Masayuki Oda , and
  • Floyd E. Romesberg*

The production of antibodies that selectively bind virtually any foreign compound is the hallmark of the immune system. While much is understood about how sequence diversity contributes to this remarkable feat of molecular recognition, little is known about how sequence diversity impacts antibody dynamics, which is also expected to contribute to molecular recognition. Toward this goal, we examined a panel of antibodies elicited to the chromophoric antigen fluorescein. On the basis of isothermal titration calorimetry, we selected six antibodies that bind fluorescein with diverse binding entropies, suggestive of varying contributions of dynamics to molecular recognition. Sequencing revealed that two pairs of antibodies employ homologous heavy chains that were derived from common germline genes, while the other two heavy chains and all six of the light chains were derived from different germline genes and are not homologous. Interestingly, more than half of all the somatic mutations acquired during affinity maturation among the six antibodies are located in positions unlikely to contact fluorescein directly. To quantify and compare the dynamics of the antibody−fluorescein complexes, three-pulse photon echo peak shift and transient grating spectroscopy were employed. All of the antibodies exhibited motions on three distinct time scales, ultrafast motions on the <100 fs time scale, diffusive motions on the picosecond time scale, and motions that occur on time scales longer than nanoseconds and thus appear static. However, the exact frequency of the picosecond time scale motion and the relative contribution of the different motions vary significantly among the antibody−chromophore complexes, revealing a high level of dynamic diversity. Using a hierarchical model, we relate the data to features of the antibodies’ energy landscapes as well as their flexibility in terms of elasticity and plasticity. In all, the data provide a consistent picture of antibody flexibility, which interestingly appears to be correlated with binding entropy as well as with germline gene use and the mutations introduced during affinity maturation. The data also provide a gauge of the dynamic diversity of the antibody repertoire and suggest that this diversity might contribute to molecular recognition by facilitating the recognition of the broadest range of foreign molecules.

Phosphorylation of Protein Phosphatase 2Cζ by c-Jun NH2-Terminal Kinase at Ser 92 Attenuates Its Phosphatase Activity
  • Kenjiro Awano ,
  • Kazutaka Amano ,
  • Yuko Nagaura ,
  • Shin-ichiro Kanno ,
  • Seishi Echigo ,
  • Shinri Tamura* , and
  • Takayasu Kobayashi*

The protein phosphatase 2C (PP2C) family represents one of the four major protein Ser/Thr phosphatase activities in mammalian cells and contains at least 13 distinct gene products. Although PP2C family members regulate a variety of cellular functions, mechanisms of regulation of their activities are largely unknown. Here, we show that PP2Cζ, a PP2C family member that is enriched in testicular germ cells, is phosphorylated by c-Jun NH2-terminal kinase (JNK) but not by p38 in vitro. Mass spectrometry and mutational analyses demonstrated that phosphorylation occurs at Ser92, Thr202, and Thr205 of PP2Cζ. Phosphorylation of these Ser and Thr residues of PP2Cζ ectopically expressed in 293 cells was enhanced by osmotic stress and was attenuated by a JNK inhibitor but not by p38 or MEK inhibitors. Phosphorylation of PP2Cζ by TAK1-activated JNK repressed its phosphatase activity in cells, and alanine mutation at Ser92 but not at Thr202 or Thr205 suppressed this inhibition. Taken together, these results suggest that specific phosphorylation of PP2Cζ at Ser92 by stress-activated JNK attenuates its phosphatase activity in cells.

Regulation of eNOS-Derived Superoxide by Endogenous Methylarginines
  • Lawrence J. Druhan ,
  • Scott P. Forbes ,
  • Arthur J. Pope ,
  • Chun-An Chen ,
  • Jay L. Zweier , and
  • Arturo J. Cardounel*

The endogenous methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine (L-NMMA) regulate nitric oxide (NO) production from endothelial NO synthase (eNOS). Under conditions of tetrahydrobiopterin (BH4) depletion eNOS also generates •O2− however, the effects of methylarginines on eNOS-derived •O2− generation are poorly understood. Therefore, using electron paramagnetic resonance spin trapping techniques we measured the dose-dependent effects of ADMA and L-NMMA on •O2− production from eNOS under conditions of BH4 depletion. In the absence of BH4, ADMA dose-dependently increased NOS-derived •O2− generation, with a maximal increase of 151% at 100 μM ADMA. L-NMMA also dose-dependently increased NOS-derived •O2−, but to a lesser extent, demonstrating a 102% increase at 100 μM L-NMMA. Moreover, the native substrate l-arginine also increased eNOS-derived •O2−, exhibiting a similar degree of enhancement as that observed with ADMA. Measurements of NADPH consumption from eNOS demonstrated that binding of either l-arginine or methylarginines increased the rate of NADPH oxidation. Spectrophotometric studies suggest, just as for l-arginine and L-NMMA, the binding of ADMA shifts the eNOS heme to the high-spin state, indicative of a more positive heme redox potential, enabling enhanced electron transfer from the reductase to the oxygenase site. These results demonstrate that the methylarginines can profoundly shift the balance of NO and •O2− generation from eNOS. These observations have important implications with regard to the therapeutic use of l-arginine and the methylarginine-NOS inhibitors in the treatment of disease.

TATA-Binding Protein Recognition and Bending of a Consensus Promoter Are Protein Species Dependent
  • JoDell E. Whittington ,
  • Roberto F. Delgadillo ,
  • Torrissa J. Attebury ,
  • Laura K. Parkhurst ,
  • Margaret A. Daugherty , and
  • Lawrence J. Parkhurst*

The structure and behavior of full-length human TBP binding the adenovirus major late promoter (AdMLP) have been characterized using biophysical methods. The human protein induces a 97° bend in DNAAdMLP. The high-resolution functional data provide a quantitative energetic and kinetic description of the partial reaction sequence as native human TBP binds rapidly to a consensus promoter with high affinity. The reaction proceeds with successive formation of three bound species, all having strongly bent DNA, with the concurrence of binding and bending demonstrated by both fluorescence and anisotropy stopped flow. These results establish the protein species dependence of the TBP−DNAAdMLP structure and recognition mechanism. Additionally, the strong correlation between the DNA bend angle and transcription efficiency demonstrated previously for yeast TBP is shown to extend to human TBP. The heterologous NH2-terminal domains are the apparent source of the species-specific differences. Together with previous studies the present work establishes that TBPwt−DNATATA function and structure depend both on the TATA box sequence and on the TBP species.

Sre1, an Iron-Modulated GATA DNA-Binding Protein of Iron-Uptake Genes in the Fungal Pathogen Histoplasma capsulatum
  • Lily Y. Chao ,
  • Michael A. Marletta* , and
  • Jasper Rine*

The pathogenic fungus Histoplasma capsulatum requires iron for its survival during macrophage infection. Because iron is toxic at high levels, iron acquisition in pathogenic organisms, including H. capsulatum, is a highly regulated process. In response to excess iron, H. capsulatum represses transcription of genes involved in iron uptake. We report here that SRE1, a gene encoding a GATA-type protein, bound to promoter sequences of genes involved in siderophore biosynthesis. Sre1 had sequence similarity to the fungal negative regulators of siderophore biosynthesis. Expression of SRE1 was reduced under iron-starving conditions, underscoring its role as a negative regulator of genes involved in iron uptake. Sre1p specifically bound DNA containing the 5′-(G/A)ATC(T/A)GATAA-3′ sequence, and that binding was both iron- and zinc-dependent. Metal analysis indicated that a substoichiometric amount of iron, predominately Fe3+, was bound to the purified protein. About 0.5−1 equiv of Fe3+ per monomer was necessary for full DNA-binding activity. Mutations in the conserved cysteine residues in the cysteine-rich region led to a decrease in bound iron. The loss of iron led to a ∼2.5-fold decrease in DNA-binding affinity, indicating that iron was directly involved in SRE1 regulation of iron-uptake genes.

RNA Binding Specificity of Drosophila Muscleblind
  • Emily S. Goers ,
  • Rodger B. Voelker ,
  • Devika P. Gates , and
  • J. Andrew Berglund*

Members of the muscleblind family of RNA binding proteins found in Drosophila and mammals are key players in both the human disease myotonic dystrophy and the regulation of alternative splicing. Recently, the mammalian muscleblind-like protein, MBNL1, has been shown to have interesting RNA binding properties with both endogenous and disease-related RNA targets. Here we report the characterization of RNA binding properties of the Drosophila muscleblind protein Mbl. Mutagenesis of double-stranded CUG repeats demonstrated that Mbl requires pyrimidine-pyrimidine mismatches for binding and that the identity and location of the C-G and G-C base pairs within the repeats are essential for Mbl binding. Systematic evolution of ligands by exponential enrichment (SELEX) was used to identify RNA sequences that bind Mbl with much higher affinity than CUG repeats. The RNA sequences identified by SELEX are structured and contain a five-nucleotide consensus sequence of 5′-AGUCU-3′. RNase footprinting of one of the SELEX RNA sequences with Mbl showed that Mbl binds both double-stranded and single-stranded regions of the RNA. Three guanosines show the strongest footprint in the presence of Mbl mutation of any of these three guanosines eliminates Mbl binding. It was also found that Mbl specifically bound a human MBNL1 RNA target, demonstrating the conservation of the muscleblind proteins in recognizing RNA targets. Our results reveal that Mbl recognizes complex RNA secondary structures.

Isotope Sensitive Branching and Kinetic Isotope Effects in the Reaction of Deuterated Arachidonic Acids with Human 12- and 15-Lipoxygenases
  • Cyril Jacquot ,
  • Aaron T. Wecksler ,
  • Chris M. McGinley ,
  • Erika N. Segraves ,
  • Theodore R. Holman* , and
  • Wilfred A. van der Donk*

Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30−80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2.

A Second Conserved GAF Domain Cysteine Is Required for the Blue/Green Photoreversibility of Cyanobacteriochrome Tlr0924 from Thermosynechococcus elongatus
  • Nathan C. Rockwell ,
  • Stephanie Lane Njuguna ,
  • Laurel Roberts ,
  • Elenor Castillo ,
  • Victoria L. Parson ,
  • Sunshine Dwojak ,
  • J. Clark Lagarias* , and
  • Susan C. Spiller*

Phytochromes are widely occurring red/far-red photoreceptors that utilize a linear tetrapyrrole (bilin) chromophore covalently bound within a knotted PAS-GAF domain pair. Cyanobacteria also contain more distant relatives of phytochromes that lack this knot, such as the phytochrome-related cyanobacteriochromes implicated to function as blue/green switchable photoreceptors. In this study, we characterize the cyanobacteriochrome Tlr0924 from the thermophilic cyanobacterium Thermosynechococcus elongatus. Full-length Tlr0924 exhibits blue/green photoconversion across a broad range of temperatures, including physiologically relevant temperatures for this organism. Spectroscopic characterization of Tlr0924 demonstrates that its green-absorbing state is in equilibrium with a labile, spectrally distinct blue-absorbing species. The photochemically generated blue-absorbing state is in equilibrium with another species absorbing at longer wavelengths, giving a total of 4 states. Cys499 is essential for this behavior, because mutagenesis of this residue results in red-absorbing mutant biliproteins. Characterization of the C499D mutant protein by absorbance and CD spectroscopy supports the conclusion that its bilin chromophore adopts a similar conformation to the red-light-absorbing Pr form of phytochrome. We propose a model photocycle in which Z/E photoisomerization of the 15/16 bond modulates formation of a reversible thioether linkage between Cys499 and C10 of the chromophore, providing the basis for the blue/green switching of cyanobacteriochromes.

Interfacial metal coordination in engineered protein and peptide assemblies

Engineered peptide scaffolds that assemble in response to metal coordination.

Design of metal-mediated protein assemblies with new structural/chemical properties.

Applications of coordination chemistry to control supramolecular protein assembly.

Metal ions are frequently found in natural protein–protein interfaces, where they stabilize quaternary or supramolecular protein structures, mediate transient protein–protein interactions, and serve as catalytic centers. Paralleling these natural roles, coordination chemistry of metal ions is being increasingly utilized in creative ways toward engineering and controlling the assembly of functional supramolecular peptide and protein architectures. Here we provide a brief overview of this emerging branch of metalloprotein/peptide engineering and highlight a few select examples from the recent literature that best capture the diversity and future potential of approaches that are being developed.

D7. Redox Chemistry and Protein Folding - Biology

4′-Phosphopantetheinyl transferases (PPTase) post-translationally modify carrier proteins with a phosphopantetheine moiety, an essential reaction in all three domains of life. In the bacterial genus Mycobacteria, the Sfp-type PPTase activates pathways necessary for the biosynthesis of cell wall components and small molecule virulence factors. We solved the X-ray crystal structures and biochemically characterized the Sfp-type PPTases from two of the most prevalent Mycobacterial pathogens, PptT of M. tuberculosis and MuPPT of M. ulcerans. Structural analyses reveal significant differences in cofactor binding and active site composition when compared to previously characterized Sfp-type PPTases. Functional analyses including the efficacy of Sfp-type PPTase-specific inhibitors also suggest that the Mycobacterial Sfp-type PPTases can serve as therapeutic targets against Mycobacterial infections.

Heat-Shock Response Transcriptional Program Enables High-Yield and High-Quality Recombinant Protein Production in Escherichia coli
  • Xin Zhang ,
  • Yu Liu ,
  • Joseph C. Genereux ,
  • Chandler Nolan ,
  • Meha Singh , and
  • Jeffery W. Kelly*

The biosynthesis of soluble, properly folded recombinant proteins in large quantities from Escherichia coli is desirable for academic research and industrial protein production. The basal E. coli protein homeostasis (proteostasis) network capacity is often insufficient to efficiently fold overexpressed proteins. Herein we demonstrate that a transcriptionally reprogrammed E. coli proteostasis network is generally superior for producing soluble, folded, and functional recombinant proteins. Reprogramming is accomplished by overexpressing a negative feedback deficient heat-shock response transcription factor before and during overexpression of the protein-of-interest. The advantage of transcriptional reprogramming versus simply overexpressing select proteostasis network components (e.g., chaperones and co-chaperones, which has been explored previously) is that a large number of proteostasis network components are upregulated at their evolved stoichiometry, thus maintaining the system capabilities of the proteostasis network that are currently incompletely understood. Transcriptional proteostasis network reprogramming mediated by stress-responsive signaling in the absence of stress should also be useful for protein production in other cells.

Functional and Structural Characterization of 2-Amino-4-phenylthiazole Inhibitors of the HIV-1 Nucleocapsid Protein with Antiviral Activity
  • Mattia Mori ,
  • Alessandro Nucci ,
  • Maria Chiara Dasso Lang ,
  • Nicolas Humbert ,
  • Christian Boudier ,
  • Francois Debaene ,
  • Sarah Sanglier-Cianferani ,
  • Marjorie Catala ,
  • Patricia Schult-Dietrich ,
  • Ursula Dietrich ,
  • Carine Tisné ,
  • Yves Mely* , and
  • Maurizio Botta*

The nucleocapsid protein (NC) is a highly conserved protein in diverse HIV-1 subtypes that plays a central role in virus replication, mainly by interacting with conserved nucleic acid sequences. NC is considered a highly profitable drug target to inhibit multiple steps in the HIV-1 life cycle with just one compound, a unique property not shown by any of the other antiretroviral classes. However, most of NC inhibitors developed so far act through an unspecific and potentially toxic mechanism (zinc ejection) and are mainly being investigated as topical microbicides. In an effort to provide specific NC inhibitors that compete for the binding of nucleic acids to NC, here we combined molecular modeling, organic synthesis, biophysical studies, NMR spectroscopy, and antiviral assays to design, synthesize, and characterize an efficient NC inhibitor endowed with antiviral activity in vitro, a desirable property for the development of efficient antiretroviral lead compounds.

Genetically Encoding an Electrophilic Amino Acid for Protein Stapling and Covalent Binding to Native Receptors
  • Xiao-Hua Chen ,
  • Zheng Xiang ,
  • Ying S. Hu ,
  • Vanessa K. Lacey ,
  • Hu Cang , and
  • Lei Wang*

Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability.

Designed BH3 Peptides with High Affinity and Specificity for Targeting Mcl-1 in Cells
  • Glenna Wink Foight ,
  • Jeremy A. Ryan ,
  • Stefano V. Gullá ,
  • Anthony Letai , and
  • Amy E. Keating*

Mcl-1 is overexpressed in many cancers and can confer resistance to cell-death signaling in refractory disease. Molecules that specifically inhibit Mcl-1 hold potential for diagnosing and disrupting Mcl-1-dependent cell survival. We selected three peptides from a yeast-surface display library that showed moderate specificity and affinity for binding to Mcl-1 over Bfl-1, Bcl-xL, Bcl-2, and Bcl-w. Specificity for Mcl-1 was improved by introducing threonine at peptide position 2e. The most specific peptide, MS1, bound Mcl-1 with 40-fold or greater specificity over four other human Bcl-2 paralogs. In BH3 profiling assays, MS1 caused depolarization in several human Mcl-1-dependent cell lines with EC50 values of ∼3 μM, contrasted with EC50 values of >100 μM for Bcl-2-, Bcl-xL-, or Bfl-1-dependent cell lines. MS1 is at least 30-fold more potent in this assay than the previously used Mcl-1 targeting reagent NoxaA BH3. These peptides can be used to detect Mcl-1 dependency in cells and provide leads for developing Mcl-1 targeting therapeutics.

Orthogonal Control of Antibacterial Activity with Light
  • Willem A. Velema ,
  • Jan Pieter van der Berg ,
  • Wiktor Szymanski ,
  • Arnold J. M. Driessen , and
  • Ben L. Feringa*

Selection of a single bacterial strain out of a mixture of microorganisms is of crucial importance in healthcare and microbiology research. Novel approaches that can externally control bacterial selection are a valuable addition to the microbiology toolbox. In this proof-of-concept, two complementary antibiotics are protected with photocleavable groups that can be orthogonally addressed with different wavelengths of light. This allows for the light-triggered selection of a single bacterial strain out of a mixture of multiple strains, by choosing the right wavelength. Further improvement toward additional orthogonally addressable antibiotics might ultimately lead to a novel methodology for bacterial selection in complex populations.

Differential Effects on Allele Selective Silencing of Mutant Huntingtin by Two Stereoisomers of α,β-Constrained Nucleic Acid
  • Michael E. Østergaard ,
  • Béatrice Gerland ,
  • Jean-Marc Escudier ,
  • Eric E. Swayze , and
  • Punit P. Seth*

We describe the effects of introducing two epimers of neutral backbone α,β-constrained nucleic acid (CNA) on the activity and allele selectivity profile of RNase H active antisense oligonucleotides (ASOs) targeting a single nucleotide polymorphism (SNP) for the treatment of Huntington’s disease (HD). ASOs modified with both isomers of α,β-CNA in the gap region showed good activity versus the mutant allele, but one isomer showed improved selectivity versus the wild-type allele. Analysis of the human RNase H cleavage patterns of α,β-CNA modified ASOs versus matched and mismatched RNA revealed that both isomers support RNase H cleavage on the RNA strand across from the site of incorporation in the ASO—an unusual observation for a neutral linkage oligonucleotide modification. Interestingly, ASOs modified with (R)- and (S)-5′-hydroxyethyl DNA (RHE and SHE respectively) formed by partial hydrolysis of the dioxaphosphorinane ring system in α,β-CNA also showed good activity versus the mutant allele but an improved selectivity profile was observed for the RHE modified ASO. Our observations further support the profiling of neutral and 5′-modified nucleic acid analogs as tools for gene silencing applications.

Enzymatic Synthesis of Polybrominated Dioxins from the Marine Environment

Polyhalogenated dibenzo-p-dioxins are arguably among the most toxic molecules known to man. In addition to anthropogenic sources, marine invertebrates also harbor polybrominated dibenzo-p-dioxins of as yet unknown biogenic origin. Here, we report that the bmp gene locus in marine bacteria, a recently characterized source of polybrominated diphenyl ethers, can also synthesize dibenzo-p-dioxins by employing different phenolic initiator molecules. Our findings also diversify the structural classes of diphenyl ethers accessed by the bmp biosynthetic pathway. This report lays the biochemical foundation of a likely biogenetic origin of dibenzo-p-dioxins present in the marine metabolome and greatly expands the toxicity potential of marine derived polyhaloganated natural products.

Nitroreductase-Activatable Morpholino Oligonucleotides for in Vivo Gene Silencing

Phosphorodiamidate morpholino oligonucleotides are widely used to interrogate gene function in whole organisms, and light-activatable derivatives can reveal spatial and temporal differences in gene activity. We describe here a new class of caged morpholino oligonucleotides that can be activated by the bacterial nitroreductase NfsB. We characterize the activation kinetics of these reagents in vitro and demonstrate their efficacy in zebrafish embryos that express NfsB either ubiquitously or in defined cell populations. In combination with transgenic organisms, such enzyme-actuated antisense tools will enable gene silencing in specific cell types, including tissues that are not amenable to optical targeting.

Chemical Reporter for Visualizing Metabolic Cross-Talk between Carbohydrate Metabolism and Protein Modification

Metabolic chemical reporters have been largely used to study posttranslational modifications. Generally, it was assumed that these reporters entered one biosynthetic pathway, resulting in labeling of one type of modification. However, because they are metabolized by cells before their addition onto proteins, metabolic chemical reporters potentially provide a unique opportunity to read-out on both modifications of interest and cellular metabolism. We report here the development of a metabolic chemical reporter 1-deoxy-N-pentynyl glucosamine (1-deoxy-GlcNAlk). This small-molecule cannot be incorporated into glycans however, treatment of mammalian cells results in labeling of a variety proteins and enables their visualization and identification. Competition of this labeling with sodium acetate and an acetyltransferase inhibitor suggests that 1-deoxy-GlcNAlk can enter the protein acetylation pathway. These results demonstrate that metabolic chemical reporters have the potential to isolate and potentially discover cross-talk between metabolic pathways in living cells.

Transformation of Human Cathelicidin LL-37 into Selective, Stable, and Potent Antimicrobial Compounds
  • Guangshun Wang* ,
  • Mark L. Hanke ,
  • Biswajit Mishra ,
  • Tamara Lushnikova ,
  • Cortney E. Heim ,
  • Vinai Chittezham Thomas ,
  • Kenneth W. Bayles , and
  • Tammy Kielian

This Letter reports a family of novel antimicrobial compounds obtained by combining peptide library screening with structure-based design. Library screening led to the identification of a human LL-37 peptide resistant to chymotrypsin. This d-amino-acid-containing peptide template was active against Escherichia coli but not methicillin-resistant Staphylococcus aureus (MRSA). It possesses a unique nonclassic amphipathic structure with hydrophobic defects. By repairing the hydrophobic defects, the peptide (17BIPHE2) gained activity against the ESKAPE pathogens, including Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species. In vitro, 17BIPHE2 could disrupt bacterial membranes and bind to DNA. In vivo, the peptide prevented staphylococcal biofilm formation in a mouse model of catheter-associated infection. Meanwhile, it boosted the innate immune response to further combat the infection. Because these peptides are potent, cell-selective, and stable to several proteases, they may be utilized to combat one or more ESKAPE pathogens.

How Many Antimicrobial Peptide Molecules Kill a Bacterium? The Case of PMAP-23
  • Daniela Roversi ,
  • Vincenzo Luca ,
  • Simone Aureli ,
  • Yoonkyung Park ,
  • Maria Luisa Mangoni , and
  • Lorenzo Stella*

Antimicrobial peptides (AMPs) kill bacteria mainly through the perturbation of their membranes and are promising compounds to fight drug resistance. Models of the mechanism of AMPs-induced membrane perturbation were developed based on experiments in liposomes, but their relevance for bacterial killing is debated. We determined the association of an analogue of the AMP PMAP-23 to Escherichia coli cells, under the same experimental conditions used to measure bactericidal activity. Killing took place only when bound peptides completely saturated bacterial membranes (106–107 bound peptides per cell), indicating that the “carpet” model for the perturbation of artificial bilayers is representative of what happens in real bacteria. This finding supports the view that, at least for this peptide, a microbicidal mechanism is possible in vivo only at micromolar total peptide concentrations. We also showed that, notwithstanding their simplicity, liposomes represent a reliable model to characterize AMPs partition in bacterial membranes.

Bioinspired Strategy for the Ribosomal Synthesis of Thioether-Bridged Macrocyclic Peptides in Bacteria

Inspired by the biosynthetic logic of lanthipeptide natural products, a new methodology was developed to direct the ribosomal synthesis of macrocyclic peptides constrained by an intramolecular thioether bond. As a first step, a robust and versatile strategy was implemented to enable the cyclization of ribosomally derived peptide sequences via a chemoselective reaction between a genetically encoded cysteine and a cysteine-reactive unnatural amino acid (O-(2-bromoethyl)-tyrosine). Combination of this approach with intein-catalyzed protein splicing furnished an efficient route to achieve the spontaneous, post-translational formation of structurally diverse macrocyclic peptides in bacterial cells. The present peptide cyclization strategy was also found to be amenable to integration with split intein-mediated circular ligation, resulting in the intracellular synthesis of conformationally constrained peptides featuring a bicyclic architecture.

Nucleophilic 1,4-Additions for Natural Product Discovery
  • Courtney L. Cox ,
  • Jonathan I. Tietz ,
  • Karol Sokolowski ,
  • Joel O. Melby ,
  • James R. Doroghazi , and
  • Douglas A. Mitchell*

Natural products remain an important source of drug candidates, but the difficulties inherent to traditional isolation, coupled with unacceptably high rates of compound rediscovery, limit the pace of natural product detection. Here we describe a reactivity-based screening method to rapidly identify exported bacterial metabolites that contain dehydrated amino acids (i.e., carbonyl- or imine-activated alkenes), a common motif in several classes of natural products. Our strategy entails the use of a commercially available thiol, dithiothreitol, for the covalent labeling of activated alkenes by nucleophilic 1,4-addition. Modification is easily discerned by comparing mass spectra of reacted and unreacted cell surface extracts. When combined with bioinformatic analysis of putative natural product gene clusters, targeted screening and isolation can be performed on a prioritized list of strains. Moreover, known compounds are easily dereplicated, effectively eliminating superfluous isolation and characterization. As a proof of principle, this labeling method was used to identify known natural products belonging to the thiopeptide, lanthipeptide, and linaridin classes. Further, upon screening a panel of only 23 actinomycetes, we discovered and characterized a novel thiopeptide antibiotic, cyclothiazomycin C.

Structures of Kibdelomycin Bound to Staphylococcus aureus GyrB and ParE Showed a Novel U-Shaped Binding Mode
  • Jun Lu* ,
  • Sangita Patel ,
  • Nandini Sharma ,
  • Stephen M. Soisson ,
  • Ryuta Kishii ,
  • Masaya Takei ,
  • Yasumichi Fukuda ,
  • Kevin J. Lumb , and
  • Sheo B. Singh*

Bacterial resistance to antibiotics continues to pose serious challenges as the discovery rate for new antibiotics fades. Kibdelomycin is one of the rare, novel, natural product antibiotics discovered recently that inhibits the bacterial DNA synthesis enzymes gyrase and topoisomerase IV. It is a broad-spectrum, Gram-positive antibiotic without cross-resistance to known gyrase inhibitors, including clinically effective quinolones. To understand its mechanism of action, binding mode, and lack of cross-resistance, we have co-crystallized kibdelomycin and novobiocin with the N-terminal domains of Staphylococcus aureus gyrase B (24 kDa) and topo IV (ParE, 24 and 43 kDa). Kibdelomycin shows a unique “dual-arm”, U-shaped binding mode in both crystal structures. The pyrrolamide moiety in the lower part of kibdelomycin penetrates deeply into the ATP-binding site pocket, whereas the isopropyl-tetramic acid and sugar moiety of the upper part thoroughly engage in polar interactions with a surface patch of the protein. The isoproramic acid (1,3-dioxopyrrolidine) and a tetrahydropyran acetate group (Sugar A) make polar contact with a surface area consisting of helix α4 and the flexible loop connecting helices α3 and α4. The two arms are connected together by a rigid decalin linker that makes van del Waals contacts with the protein backbone. This “dual-arm”, U-shaped, multicontact binding mode of kibdelomycin is unique and distinctively different from binding modes of other known gyrase inhibitors (e.g., coumarins and quinolones), which explains its lack of cross-resistance and low frequency of resistance. The crystal structures reported in this paper should enable design and discovery of analogues with better properties and antibacterial spectrum.

Alterations in Energy/Redox Metabolism Induced by Mitochondrial and Environmental Toxins: A Specific Role for Glucose-6-Phosphate-Dehydrogenase and the Pentose Phosphate Pathway in Paraquat Toxicity
  • Shulei Lei ,
  • Laura Zavala-Flores ,
  • Aracely Garcia-Garcia ,
  • Renu Nandakumar ,
  • Yuting Huang ,
  • Nandakumar Madayiputhiya ,
  • Robert C. Stanton ,
  • Eric D. Dodds ,
  • Robert Powers* , and
  • Rodrigo Franco*

Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression.

Mia40 Is Optimized for Function in Mitochondrial Oxidative Protein Folding and Import

Mia40 catalyzes oxidative protein folding in mitochondria. It contains a unique catalytic CPC dithiol flanked by a hydrophobic groove, and unlike other oxidoreductases, it forms long-lived mixed disulfides with substrates. We show that this distinctive property originates neither from particular properties of mitochondrial substrates nor from the CPC motif of Mia40. The catalytic cysteines of Mia40 display unusually low chemical reactivity, as expressed in conventional pK values and reduction potentials. The stability of the mixed disulfide intermediate is coupled energetically with hydrophobic interactions between Mia40 and the substrate. Based on these properties, we suggest a mechanism for Mia40, where the hydrophobic binding site is employed to select a substrate thiol for forming the initial mixed disulfide. Its long lifetime is used to retain partially folded proteins in the mitochondria and to direct folding toward forming the native disulfide bonds.


The use of side chains as catalytic cofactors for protein mediated redox chemistry raises significant mechanistic issues as to how these amino acids are activated toward radical chemistry in a controlled manner. De novo protein design has been used to examine the structural basis for the creation and maintenance of a tryptophanyl radical in a three-helix bundle protein maquette. Here we report the detailed structural analysis of the protein by multidimensional NMR methods. An interesting feature of the structure is an apparent π-cation interaction involving the sole tryptophan and a lysine side chain. Hybrid density functional calculations support the notion that this interaction raises the reduction potential of the W°/WH redox pair and helps explain the redox characteristics of the protein. This model protein system therefore provides a powerful model for exploring the structural basis for controlled radical chemistry in protein.

Watch the video: 8. Protein Folding 1 (May 2022).