Structural basis for ubiquitin recognition by SH3 domains

The SH3 domain is a protein-protein interaction module commonly found in intracellular signaling and adaptor proteins. The SH3 domains of multiple endocytic proteins have been recently implicated in binding ubiquitin, which serves as a signal for diverse cellular processes including gene regulation, endosomal sorting, and protein destruction. Here we describe the solution NMR structure of ubiquitin in complex with an SH3 domain belonging to the yeast endocytic protein Sla1. The ubiquitin binding surface of the Sla1 SH3 domain overlaps substantially with the canonical binding surface for proline-rich ligands. Like many other ubiquitin-binding motifs, the SH3 domain engages the Ile44 hydrophobic patch of ubiquitin. A phenylalanine residue located at the heart of the ubiquitin-binding surface of the SH3 domain serves as a key specificity determinant. The structure of the SH3-ubiquitin complex explains how a subset of SH3 domains has acquired this non-traditional function.     

The Effects of CapZ Peptide (TRTK-12) Binding to S100B-Ca as Examined by NMR and X-ray Crystallography

Structure-based drug design is underway to inhibit the S100B-p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between Ca²⁺-S100B and TRTK-12, a target that binds to the p53-binding site on S100B. The structures of Ca²⁺-S100B (1.5-Å resolution) and S100B-Ca²⁺-TRTK-12 (2.0-Å resolution) determined here indicate that the S100B-Ca²⁺-TRTK-12 complex is dominated by an interaction between Trp7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca²⁺-S100B involving residues in helices 2 and 3 and loop 2. As with an S100B-Ca²⁺-p53 peptide complex, TRTK-12 binding to Ca²⁺-S100B was found to increase the protein's Ca²⁺-binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca²⁺ ligands and/or improved the Ca²⁺ coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca²⁺-TRTK-12 and S100B-Ca²⁺ were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD = 0.19). On the other hand, B-factors for residues in EF2 of Ca²⁺-S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR ¹⁵N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca²⁺-S100B. Such a loss of protein motion may also provide an explanation for how calcium-ion-binding affinity is increased upon binding a target. Lastly, it follows that any small-molecule inhibitor bound to Ca²⁺-S100B would also have to cause an increase in calcium-ion-binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug design studies involving S100B.     

The mycobacterial Mpa–proteasome unfolds and degrades pupylated substrates by engaging Pup's N‐terminus

Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin‐like protein (Pup) that is recognized by the N‐terminal coiled‐coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa–proteasome complex unfolds and degrades Pup‐tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N‐terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.     

Structure of the leech protein saratin and characterization of its binding to collagen

The leech protein Saratin from Hirudo medicinalis prevents thrombocyte aggregation by interfering with the first binding step of the thrombocytes to collagen by binding to collagen. We solved the three-dimensional structure of the leech protein Saratin in solution and identified its collagen binding site by NMR titration experiments. The NMR structure of Saratin consists of one α-helix and a five-stranded β-sheet arranged in the topology ββαβββ. The C-terminal region, of about 20 amino acids in length, adopts no regular structure. NMR titration experiments with collagen peptides show that the collagen interaction of Saratin takes place in a kind of notch that is formed by the end of the α-helix and the β-sheet. NMR data-driven docking experiments to collagen model peptides were used to elucidate the putative binding mode of Saratin and collagen. Mainly, parts of the first and the end of the fifth β-strand, the loop connecting the α-helix and the third β-strand, and a short part of the loop connecting the fourth and fifth β-strand participate in binding.     

Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation

Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.     

A hydrogen bond regulates slow motions in ubiquitin by modulating a β-turn flip

Proteins exist as conformational ensembles composed of multiple interchanging substates separated by kinetic barriers. Interconverting conformations are often difficult to probe, owing to their sparse population and transient nature. Here, we report the identification and characterization of a subset of conformations in ubiquitin that participate in microsecond-to-millisecond motions in the amides of Ile23, Asn25, and Thr55. A novel side chain to the backbone hydrogen bond that regulates these motions has also been identified. Combining our NMR studies with the available X-ray data, we have unearthed the physical process underlying slow motions—the interconversion of a type I into a type II β-turn flip at residues Glu51 through Arg54. Interestingly, the dominant conformer of wild-type ubiquitin observed in solution near neutral pH is only represented by about 22% of the crystal structures. The conformers generated as a result of the dynamics of the hydrogen bond appear to be correlated to ligand recognition by ubiquitin.     

Molecular basis of Bcl-xL's target recognition versatility revealed by the structure of Bcl-xL in complex with the BH3 domain of Beclin-1

Beclin-1, originally identified as a Bcl-2 binding protein, is an evolutionarily conserved protein required for autophagy. The direct interaction between Beclin-1 and Bcl-2 or Bcl-xL provides a potential convergence point for apoptosis and autophagy, two programmed cell death processes. Given the functional significance of the interaction between Beclin-1 and Bcl-2/Bcl-xL, we performed detailed biochemical and structural characterizations of this interaction. We demonstrated that the Bcl-xL-binding domain of Beclin-1 contains a BH3 domain. Therefore, Beclin-1 is a new member of the BH3-only family proteins. The structure of Bcl-xL in complex with the Beclin-1 BH3 domain was determined at high resolution by NMR spectroscopy. Although similar to other known BH3 domains, the Beclin-1 BH3 domain displays its own distinct features in the complex with Bcl-xL. Systematic analysis of all known Bcl-xL/BH3 domain complexes helped us to identify the molecular basis underlying the capacity of Bcl-xL to recognize diverse target sequences.     

Electrostatic contributions to the stability of the GCN4 leucine zipper structure

Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK_a values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK_a values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pK_a values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pK_a differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pK_a values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pK_a predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.     

NMR structure of the Escherichia coli type 1 pilus subunit FimF and its interactions with other pilus subunits

Type 1 pili from uropathogenic Escherichia coli strains mediate bacterial attachment to target receptors on the host tissue. They are composed of up to 3000 copies of the subunit FimA, which form the stiff, helical pilus rod, and the subunits FimF, FimG, and FimH, which form the linear tip fibrillum. All subunits in the pilus interact via donor strand complementation, in which the incomplete immunoglobulin-like fold of each subunit is complemented by insertion of an N-terminal extension from the following subunit. We determined the NMR structure of a monomeric, self-complemented variant of FimF, FimF_F, which has a second FimF donor strand segment fused to its C-terminus that enables intramolecular complementation of the FimF fold. NMR studies on bimolecular complexes between FimF_F and donor strand-depleted variants of FimF and FimG revealed that the relative orientations of neighboring domains in the tip fibrillum cover a wide range. The data provide strong support for the intrinsic flexibility of the tip fibrillum. They lend further support to the hypothesis that this flexibility would significantly increase the probability that the adhesin at the distal end of the fibrillum successfully targets host cell receptors.     

Conformational Dynamics and Structural Plasticity Play Critical Roles in the Ubiquitin Recognition of a UIM Domain

Ubiquitin-interacting motifs (UIMs) are an important class of protein domains that interact with ubiquitin or ubiquitin-like proteins. These approximately 20-residue-long domains are found in a variety of ubiquitin receptor proteins and serve as recognition modules towards intracellular targets, which may be individual ubiquitin subunits or polyubiquitin chains attached to a variety of proteins. Previous structural studies of interactions between UIMs and ubiquitin have shown that UIMs adopt an extended structure of a single α-helix, containing a hydrophobic surface with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin. In light of this large body of structural studies, details regarding the presence and the roles of structural dynamics and plasticity are surprisingly lacking. In order to better understand the structural basis of ubiquitin-UIM recognition, we have characterized changes in the structure and dynamics of ubiquitin upon binding of a UIM domain from the yeast Vps27 protein. The solution structure of a ubiquitin-UIM fusion protein designed to study these interactions is reported here and found to consist of a well-defined ubiquitin core and a bipartite UIM helix. Moreover, we have studied the plasticity of the docking interface, as well as global changes in ubiquitin due to UIM binding at the picoseconds-to-nanoseconds and microseconds-to-milliseconds protein motions by nuclear magnetic resonance relaxation. Changes in generalized-order parameters of amide groups show a distinct trend towards increased structural rigidity at the UIM-ubiquitin interface relative to values determined in unbound ubiquitin. Analysis of ¹⁵N Carr-Purcell-Meiboom-Gill relaxation dispersion measurements suggests the presence of two types of motions: one directly related to the UIM-binding interface and the other induced to distal parts of the protein. This study demonstrates a case where localized interactions among protein domains have global effects on protein motions at timescales ranging from picoseconds to milliseconds.     

Activity of the mycobacterial proteasomal ATPase Mpa is reversibly regulated by pupylation

Pupylation is a bacterial post-translational modification of target proteins on lysine residues with prokaryotic ubiquitin-like protein Pup. Pup-tagged substrates are recognized by a proteasome-interacting ATPase termed Mpa in Mycobacterium tuberculosis. Mpa unfolds pupylated substrates and threads them into the proteasome core particle for degradation. Interestingly, Mpa itself is also a pupylation target. Here, we show that the Pup ligase PafA predominantly produces monopupylated Mpa modified homogeneously on a single lysine residue within its C-terminal region. We demonstrate that this modification renders Mpa functionally inactive. Pupylated Mpa can no longer support Pup-mediated proteasomal degradation due to its inability to associate with the proteasome core. Mpa is further inactivated by rapid Pup- and ATPase-driven deoligomerization of the hexameric Mpa ring. We show that pupylation of Mpa is chemically and functionally reversible. Mpa regains its enzymatic activity upon depupylation by the depupylase Dop, affording a rapid and reversible activity control over Mpa function.     

A distinct structural region of the prokaryotic ubiquitin-like protein (Pup) is recognized by the N-terminal domain of the proteasomal ATPase Mpa

The mycobacterial ubiquitin-like protein Pup is coupled to proteins, thereby rendering them as substrates for proteasome-mediated degradation. The Pup-tagged proteins are recruited by the proteasomal ATPase Mpa (also called ARC). Using a combination of biochemical and NMR methods, we characterize the structural determinants of Pup and its interaction with Mpa, demonstrating that Pup adopts a range of extended conformations with a short helical stretch in its C-terminal portion. We show that the N-terminal coiled-coil domain of Mpa makes extensive contacts along the central region of Pup leaving its N-terminus unconstrained and available for other functional interactions.

Structured summary

MINT-7262427: pup (uniprotkb:B6DAC1) binds (MI:0407) to mpa (uniprotkb:Q0G9Y7) by pull down (MI:0096) MINT-7262440: mpa (uniprotkb:Q0G9Y7) and pup (uniprotkb:B6DAC1) bind (MI:0407) by isothermal titration calorimetry (MI:0065)     

Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface

H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that binds to H/ACA RNAs specifically. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent from eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of Saccharomyces cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10 and RNA binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs expressed in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA.     

NMR Structure of a Monomeric Intermediate on the Evolutionarily Optimized Assembly Pathway of a Small Trimerization Domain

Efficient formation of specific intermolecular interactions is essential for self-assembly of biological structures. The foldon domain is an evolutionarily optimized trimerization module required for assembly of the large, trimeric structural protein fibritin from phage T4. Monomers consisting of the 27 amino acids comprising a single foldon domain subunit spontaneously form a natively folded trimer. During assembly of the foldon domain, a monomeric intermediate is formed on the submillisecond time scale, which provides the basis for two consecutive very fast association reactions. Mutation of an intermolecular salt bridge leads to a monomeric protein that resembles the kinetic intermediate in its spectroscopic properties. NMR spectroscopy revealed essentially native topology of the monomeric intermediate with defined hydrogen bonds and side-chain interactions but largely reduced stability compared to the native trimer. This structural preorganization leads to an asymmetric charge distribution on the surface that can direct rapid subunit recognition. The low stability of the intermediate allows a large free-energy gain upon trimerization, which serves as driving force for rapid assembly. These results indicate different free-energy landscapes for folding of small oligomeric proteins compared to monomeric proteins, which typically avoid the transient population of intermediates.     

Structural Basis of the Regulation of the CbpA Co-chaperone by its Specific Modulator CbpM

CbpA, one of the Escherichia coli DnaJ homologues, acts as a co-chaperone in the DnaK chaperone system. Despite its extensive similarity in domain structure and function to DnaJ, CbpA has a unique and specific regulatory mechanism mediated through the small protein CbpM. Both CbpA and CbpM are highly conserved in bacteria. Earlier studies showed that CbpM interacts with the N-terminal J-domain of CbpA inhibiting its co-chaperone activity but the structural basis of this interaction is not known. Here, we have combined NMR spectroscopy, site-directed mutagenesis and surface plasmon resonance to characterize the CbpA/CbpM interaction at the molecular level. We have determined the solution structure of the CbpA J-domain and mapped the residues that are perturbed upon CbpM binding. The NMR data defined a broad region on helices α2 and α3 as involved in the interactions. Site-directed mutagenesis has been used to further delineate the CbpA J-domain/CbpM interface. We show that the binding sites of CbpM and DnaK on CbpA J-domain overlap, which suggests a competition between DnaK and CbpM for binding to CbpA as a mechanism for CbpA regulation. This study also provides the explanation for the specificity of CbpM for CbpA versus DnaJ, by identifying the key residues for differential binding.     

Design and Structure of an Equilibrium Protein Folding Intermediate: A Hint into Dynamical Regions of Proteins

Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity.     

Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins

Background

The post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa. While the disordered behavior of free Pup has been well characterized, it remained unknown whether Pup adopts a distinct structure when attached to a substrate.

Results

Using a combination of NMR experiments and biochemical analysis we demonstrate that Pup remains unstructured when ligated to two well-established pupylation substrates targeted for proteasomal degradation in Mycobacterium tuberculosis, malonyl transacylase (FabD) and ketopantoyl hydroxylmethyltransferase (PanB). Isotopically labeled Pup was linked to FabD and PanB by in vitro pupylation to generate homogeneously pupylated substrates suitable for NMR analysis. The single target lysine of PanB was identified by a combination of mass spectroscopy and mutational analysis. Chemical shift comparison between Pup in its free form and ligated to substrate reveals intrinsic disorder of Pup in the conjugate.

Conclusion

When linked to the proteasomal substrates FabD and PanB, Pup is unstructured and retains the ability to interact with its different binding partners. This suggests that it is not the conformation of Pup attached to these two substrates which determines their delivery to the proteasome, but the availability of the degradation complex and the depupylase.

     

Mapping the interactions between Lys48 and Lys63-linked di-ubiquitins and a ubiquitin-interacting motif of S5a

Numerous cellular processes are regulated by (poly)ubiquitin-mediated signaling events, which involve a covalent modification of the substrate protein by a single ubiquitin or a chain of ubiquitin molecules linked via a specific lysine. Remarkably, the outcome of polyubiquitination is linkage-dependent. For example, Lys48-linked chains are the principal signal for proteasomal degradation, while Lys63-linked chains act as nonproteolytic signals. Despite significant progress in characterization of various cellular pathways involving ubiquitin, understanding of the structural details of polyubiquitin chain recognition by downstream cellular effectors is missing. Here we use NMR to study the interaction of a ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a with di-ubiquitin, the simplest model for polyubiquitin chain, to gain insights into the mechanism of polyubiquitin recognition by the proteasome. We have mapped the binding interface and characterized the stoichiometry and the process of UIM binding to Lys48- and Lys63-linked di-ubiquitin chains. Our data provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interdomain interface. This allows UIM to enter the interface and bind directly to the same ubiquitin hydrophobic-patch surface as utilized in UIM:monoubiquitin complexes. The results indicate that up to two UIM molecules can bind di-ubiquitin, and the binding interface between UIM and ubiquitin units in di-ubiquitin is essentially the same for both Lys48- and Lys63-linked chains. Our data suggest possible structural models for the binding of UIM and of full-length S5a to di-ubiquitin.     

Dynamic conformational equilibria in the physiological function of the Bombyx mori pheromone-binding protein

The Bombyx mori pheromone-binding protein (BmorPBP) undergoes a pH-dependent conformational transition from a form at basic pH, which contains an open cavity suitable for ligand binding (BmorPBP^B), to a form at pH 4.5, where this cavity is occupied by an additional helix (BmorPBP^A). This helix α7 is formed by the C-terminal dodecapeptide 131-142, which is flexibly disordered on the protein surface in BmorPBP^B and in its complex with the pheromone bombykol. Previous work showed that the ligand-binding cavity cannot accommodate both bombykol and helix α7. Here we further investigated mechanistic aspects of the physiologically crucial ejection of the ligand at lower pH values by solution NMR studies of the variant protein BmorPBP(1-128), where the C-terminal helix-forming tetradecapeptide is removed. The NMR structure of the truncated protein at pH 6.5 corresponds closely to BmorPBP^B. At pH 4.5, BmorPBP(1-128) maintains a B-type structure that is in a slow equilibrium, on the NMR chemical shift timescale, with a low-pH conformation for which a discrete set of ¹⁵N-¹H correlation peaks is NMR unobservable. The full NMR spectrum was recovered upon readjusting the pH of the protein solution to 6.5. These data reveal dual roles for the C-terminal tetradecapeptide of BmorPBP in the mechanism of reversible pheromone binding and transport, where it governs dynamic equilibria between two locally different protein conformations at acidic pH and competes with the ligand for binding to the interior cavity.     

Allochromatium vinosum DsrC: solution-state NMR structure, redox properties, and interaction with DsrEFH, a protein essential for purple sulfur bacterial sulfur oxidation

Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and we describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that, in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. While an intermolecular Cys111-Cys111 disulfide-bonded dimer is rapidly formed under oxidizing conditions, the intramolecularly disulfide-bonded species (Cys100-Cys111) is the thermodynamically stable form of the protein under these conditions. Treatment of the disulfidic forms with reducing agent regenerates the monomeric species that was structurally characterized. Using a band-shift technique under nondenaturing conditions, we obtained evidence for the interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form, this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and are consistent with a role as a sulfur-substrate binding/transferring protein, as well as with an electron-transfer function via thiol-disulfide interchanges.