Hydroxynitrile lyase from Hevea brasiliensis: Molecular characterization and mechanism of enzyme catalysis

(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis is a 29 kDa single chain protein that catalyses the breakdown or formation of a C(SINGLE BOND)C bond by reversible addition of hydrocyanic acid to aldehydes or ketones. The primary sequence of Hnl has no significant homology to known proteins. Detailed homology investigations employing PROFILESEARCH and secondary structure prediction algorithms suggest that Hnl is a member of the α/β hydrolase fold protein family and contains a catalytic triad as functional residues for catalysis. The significance of the predicted catalytic residues was tested and confirmed by site-directed mutagenesis and expression of mutant and wild-type proteins in the yeast, Saccharomyces cerevisiae. Based on these data we suggest a mechanistic model for the (S)-cyanohydrin synthesis catalyzed by hydroxynitrile lyase from Hevea brasiliensis. Proteins 27:438–449, 1997. © 1997 Wiley-Liss, Inc.     

Disulfide bonds in ER protein folding and homeostasis

Proteins that are expressed outside the cell must be synthesized, folded, and assembled in a way that ensures they can function in their designate location. Accordingly, these proteins are primarily synthesized in the endoplasmic reticulum (ER), which has developed a chemical environment more similar to that outside the cell. This organelle is equipped with a variety of molecular chaperones and folding enzymes that both assist the folding process, while at the same time exerting tight quality control measures that are largely absent outside the cell. A major post-translational modification of ER-synthesized proteins is disulfide bridge formation, which is catalyzed by the family of protein disulfide isomerases. As this covalent modification provides unique structural advantages to extracellular proteins, multiple pathways to disulfide bond formation have evolved. However, the advantages that disulfide bonds impart to these proteins come at a high cost to the cell. Very recent reports have shed light on how the cell can deal with or even exploit the side reactions of disulfide bond formation to maintain homeostasis of the ER and its folding machinery.     

Thioredoxins and glutaredoxins as facilitators of protein folding

Thiol-disulfide oxidoreductase systems of bacterial cytoplasm and eukaryotic cytosol favor reducing conditions and protein thiol groups, while bacterial periplasm and eukaryotic endoplasmatic reticulum provide oxidizing conditions and a machinery for disulfide bond formation in the secretory pathway. Oxidoreductases of the thioredoxin fold superfamily catalyze steps in oxidative protein folding via protein-protein interactions and covalent catalysis to act as chaperones and isomerases of disulfides to generate a native fold. The active site dithiol/disulfide of thioredoxin fold proteins is CXXC where variations of the residues inside the disulfide ring are known to increase the redox potential like in protein disulfide isomerases. In the catalytic mechanism thioredoxin fold proteins bind to target proteins through conserved backbone-backbone hydrogen bonds and induce conformational changes of the target disulfide followed by nucleophilic attack by the N-terminally located low pK(a) Cys residue. This generates a mixed disulfide covalent bond which subsequently is resolved by attack from the C-terminally located Cys residue. This review will focus on two members of the thioredoxin superfamily of proteins known to be crucial for maintaining a reduced intracellular redox state, thioredoxin and glutaredoxin, and their potential functions as facilitators and regulators of protein folding and chaperone activity.     

Geometric characteristics of hydrogen bonds involving sulfur atoms in proteins

Sulfur atoms have been known to participate in hydrogen bonds (H‐bonds) and these sulfur‐containing H‐bonds (SCHBs) are suggested to play important roles in certain biological processes. This study aims to comprehensively characterize all the SCHBs in 500 high‐resolution protein structures (≤1.8 Å). We categorized SCHBs into six types according to donor/acceptor behaviors and used explicit hydrogen approach to distinguish SCHBs from those of nonhydrogen bonding interactions. It is revealed that sulfur atom is a very poor H‐bond acceptor, but a moderately good H‐bond donor. In α‐helix, considerable SCHBs were found between the sulphydryl group of cysteine residue i and the carbonyl oxygen of residue i‐4, and these SCHBs exert effects in stabilizing helices. Although for other SCHBs, they possess no specific secondary structural preference, their geometric characteristics in proteins and in free small compounds are significantly distinct, indicating the protein SCHBs are geometrically distorted. Interestingly, sulfur atom in the disulfide bond tends to form bifurcated H‐bond whereas in cysteine‐cysteine pairs prefer to form dual H‐bond. These special H‐bonds remarkably boost the interaction between H‐bond donor and acceptor. By oxidation/reduction manner, the mutual transformation between the dual H‐bonds and disulfide bonds for cysteine‐cysteine pairs can accurately adjust the structural stability and biological function of proteins in different environments. Furthermore, few loose H‐bonds were observed to form between the sulphydryl groups and aromatic rings, and in these cases the donor H is almost over against the rim rather than the center of the aromatic ring. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Sulfation, the up-and-coming post-translational modification: its role and mechanism in protein-protein interaction

Tyrosine sulfation is a post-translational modification entailing covalent attachment of sulfate to tyrosine residues. It takes place in the trans-Golgi, is necessary for the bioactivity of some proteins, and improves their ability to interact with other proteins. In the present work, we show that a protein containing a sulfated tyrosine with a delocalized negative charge forms a salt bridge with another protein if it has two or more adjacent arginine residues containing positive delocalized charges. These noncovalent complexes are so stable that, when submitted to collision induced dissociation, the peptides forming the complex dissociate. Just one covalent bond fragments, the covalent bond between the tyrosine oxygen and the SO3 sulfur, and is represented by the appearance of a new peak (basic peptide + SO3), suggesting that in some instances covalent bonds will break down before the noncovalent bonds between the arginine guanidinium and SO3 dissociate. The data implies that the dissociation pathway is preferred; however, fragmentation between tyrosine and the sulfate residue is a major pathway.     

Protein flexibility using constraints from molecular dynamics simulations

Proteins are held together in the native state by hydrophobic interactions, hydrogen bonds and interactions with the surrounding water, whose strength as well as spatial and temporal distribution affects protein flexibility and hence function. We study these effects using 10 ns molecular dynamics simulations of pure water and of two proteins, the glutamate receptor ligand binding domain and barnase. We find that most of the noncovalent interactions flicker on and off over typically nanoseconds, and so we can obtain good statistics from the molecular dynamics simulations. Based on this information, a topological network of rigid bonds corresponding to a protein structure with covalent and noncovalent bonds is constructed, with account being taken of the influence of the flickering hydrogen bonds. We define the duty cycle for the noncovalent interactions as the percentage of time a given interaction is present, which we use as an input to investigate flexibility/rigidity patterns, in the algorithm FIRST which constructs and analyses topological networks.     

The extracellular calcium-sensing receptor dimerizes through multiple types of intermolecular interactions

Recent studies have shown that the G protein-coupled, extracellular calcium ([Ca(2+)](o))-sensing receptor (CaR) forms disulfide-linked dimers through cysteine residues within its extracellular domain and that dimerization of the CaR has functional implications. In this study, we have investigated which of these disulfide linkages are essential for dimerization of the CaR and whether they are required for these functional interactions. Our results confirm the key roles of Cys(129) and Cys(131) in CaR dimerization. However, utilizing cross-linking of the CaR or immunoprecipitation of a non-FLAG-tagged CaR with a FLAG-tagged CaR using anti-FLAG antibody, we demonstrate that CaRs with or without these two cysteines form dimers on the cell surface to a similar extent. In addition, reconstitution of CaR-mediated signaling by cotransfection of two individually inactive mutant CaRs is nearly identical in the presence or absence of both Cys(129) and Cys(131), showing that covalent linkage of CaR dimers is not needed for functional interactions between CaR monomers. These findings suggest that the CaR has at least two distinct types of motifs mediating dimerization and functional interactions, i.e. covalent interactions involving intermolecular disulfide bonds and noncovalent, possibly hydrophobic, interactions.     

Predicting disulfide bond connectivity in proteins by correlated mutations analysis

MOTIVATION: Prediction of disulfide bond connectivity facilitates structural and functional annotation of proteins. Previous studies suggest that cysteines of a disulfide bond mutate in a correlated manner. RESULTS: We developed a method that analyzes correlated mutation patterns in multiple sequence alignments in order to predict disulfide bond connectivity. Proteins with known experimental structures and varying numbers of disulfide bonds, and that spanned various evolutionary distances, were aligned. We observed frequent variation of disulfide bond connectivity within members of the same protein families, and it was also observed that in 99% of the cases, cysteine pairs forming non-conserved disulfide bonds mutated in concert. Our data support the notion that substitution of a cysteine in a disulfide bond prompts the substitution of its cysteine partner and that oxidized cysteines appear in pairs. The method we developed predicts disulfide bond connectivity patterns with accuracies of 73, 69 and 61% for proteins with two, three and four disulfide bonds, respectively.     

Disulfide bond configuration of human cytomegalovirus glycoprotein B

Glycoprotein B (gB) is the most highly conserved of the envelope glycoproteins of human herpesviruses. The gB protein of human cytomegalovirus (CMV) serves multiple roles in the life cycle of the virus. To investigate structural properties of gB that give rise to its function, we sought to determine the disulfide bond arrangement of gB. To this end, a recombinant form of gB (gB-S) comprising the entire ectodomain of the glycoprotein (amino acids 1 to 750) was constructed and expressed in insect cells. Proteolytic fragmentation and mass spectrometry were performed using purified gB-S, and the five disulfide bonds that link 10 of the 11 highly conserved cysteine residues of gB were mapped. These bonds are C94-C550, C111-C506, C246-C250, C344-C391, and C573-C610. This configuration closely parallels the disulfide bond configuration of herpes simplex type 2 (HSV-2) gB (N. Norais, D. Tang, S. Kaur, S. H. Chamberlain, F. R. Masiarz, R. L. Burke, and F. Markus, J. Virol. 70:7379-7387, 1996). However, despite the high degree of conservation of cysteine residues between CMV gB and HSV-2 gB, the disulfide bond arrangements of the two homologs are not identical. We detected a disulfide bond between the conserved cysteine residue 246 and the nonconserved cysteine residue 250 of CMV gB. We hypothesize that this disulfide bond stabilizes a tight loop in the amino-terminal fragment of CMV gB that does not exist in HSV-2 gB. We predicted that the cysteine residue not found in a disulfide bond of CMV gB, cysteine residue 185, would play a role in dimerization, but a cysteine substitution mutant in cysteine residue 185 showed no apparent defect in the ability to form dimers. These results indicate that gB oligomerization involves additional interactions other than a single disulfide bond. This work represents the second reported disulfide bond structure for a herpesvirus gB homolog, and the discovery that the two structures are not identical underscores the importance of empirically determining structures even for highly conserved proteins.     

Functional rescue of the nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutants G185C and R202C by a second site suppressor mutation

Mutations in the gene of the G protein-coupled vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the receptor introduce additional cysteine residues. Several groups have proposed that these residues might disrupt the conserved disulfide bond of the V2 receptor. To test this hypothesis, we first calculated a structure model of the extracellular receptor domains. The model suggests that the additional cysteine residues may form a second disulfide bond with the free, nonconserved extracellular cysteine residue Cys-195 rather than impairing the conserved bond. To address this question experimentally, we used the NDI-causing mutant receptors G185C and R202C. Their Cys-195 residues were replaced by alanine to eliminate the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggesting that the second disulfide bonds are indeed formed. Furthermore we show that residue Cys-195, which is sensitive to "additional cysteine" mutations, is not conserved among the V2 receptors of other species and that the presence of an uneven number of extracellular cysteine residues, as in the human V2 receptor, is rare among class I G protein-coupled receptors.     

Disulfide-linked integrase oligomers involving C280 residues are formed in vitro and in vivo but are not essential for human immunodeficiency virus replication

The human immunodeficiency virus type 1 integrase (IN) forms an oligomer that integrates both ends of the viral DNA. The nature of the active oligomer is unclear. Recombinant IN obtained under reducing conditions is always in the form of noncovalent oligomers. However, disulfide-linked oligomers of IN were recently observed within viral particles. We show that IN produced from a baculovirus expression system can form disulfide-linked oligomers. We investigated which residues are responsible for the disulfide bridges and the relationship between the ability to form covalent dimers and IN activity. Only the mutation of residue C280 was sufficient to prevent the formation of intermolecular disulfide bridges in oligomers of recombinant IN. IN activity was studied under and versus nonreducing conditions: the formation of disulfide bridges was not required for the in vitro activities of the enzyme. Moreover, the covalent dimer does not dissociate into individual protomers on disulfide bridge reduction. Instead, IN undergoes a spontaneous multimerization process that yields a homogenous noncovalent tetramer. The C280S mutation also completely abolished the formation of disulfide bonds in the context of the viral particle. Finally, the replication of the mutant virus was investigated in replicating and arrested cells. The infectivity of the virus was not affected by the C280S IN mutation in either dividing or nondividing cells. The disulfide-linked form of the IN oligomers observed in the viral particles is thus not required for viral replication.     

Identification and characterization of disulfide bonds in proteins and peptides from tandem MS data by use of the MassMatrix MS/MS search engine

A new database search algorithm has been developed to identify disulfide-linked peptides in tandem MS data sets. The algorithm is included in the newly developed tandem MS database search program, MassMatrix. The algorithm exploits the probabilistic scoring model in MassMatrix to achieve identification of disulfide bonds in proteins and peptides. Proteins and peptides with disulfide bonds can be identified with high confidence without chemical reduction or other derivatization. The approach was tested on peptide and protein standards with known disulfide bonds. All disulfide bonds in the standard set were identified by MassMatrix. The algorithm was further tested on bovine pancreatic ribonuclease A (RNaseA). The 4 native disulfide bonds in RNaseA were detected by MassMatrix with multiple validated peptide matches for each disulfide bond with high statistical scores. Fifteen nonnative disulfide bonds were also observed in the protein digest under basic conditions (pH = 8.0) due to disulfide bond interchange. After minimizing the disulfide bond interchange (pH = 6.0) during digestion, only one nonnative disulfide bond was observed. The MassMatrix algorithm offers an additional approach for the discovery of disulfide bond from tandem mass spectrometry data.     

The contribution of cross-links to protein stability: A normal mode analysis of the configurational entropy of the native state

The vibrational entropy of native BPTI, with three disulfide bonds, was determined by use of normal mode calculations and compared with that of folded variants having either one less disulfide bond or lacking a peptide bond at the trypsin-reactive site. Favorable contributions to the free energy of 2.5–5.1 kcal/mol at 300 K were calculated for the reduction of disulfide bonds in the folded state, whereas no favorable contribution was found for the hydrolysis of the peptide bond cleaved by trypsin. This is on the order of the effect of disulfides in the unfolded state. The implications of these results for the stabilization of a folded protein by the introduction of crosslinks are discussed. © 1993 Wiley-Liss, Inc.     

Proposed mechanisms for binding of apo[a] kringle type 9 to apo B-100 in human lipoprotein[a].

The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue in apo B-100. Our fluorescence-labeling experiments and molecular modeling studies have provided evidence for possible interactions between this apo[a] kringle type and apo B-100. The fluorescent probe, fluorescein-5-maleimide, was used in parallel experiments to label free sulfhydryl moieties in lipoprotein[a] and low-density lipoprotein (LDL). In apo B-100 of LDL, Cys3734 was labeled with the probe, but this site was not labeled in autologous lipoprotein[a]. The result strongly implicates Cys3734 of apo B-100 as the residue forming the disulfide linkage with Cys4057 of apo[a]. To explore possible noncovalent interactions between apo B-100 and apo[a], the crystallographic coordinates for plasminogen kringle 4 were used to generate molecular models of the apo[a] kringle-repeat sequence (3991-4068, LPaK9), the only plasminogen kringle 4 type repeat in apo[a] having an extra cysteine residue not involved in an intramolecular disulfide bond. The Cys4057 residue (henceforth designated as Cys67 in the LPaK9 sequence) is believed to form an intermolecular disulfide bond with a cysteine of apo B-100. In computer graphics molecular models of LPaK9, Cys67 is located on the surface of the kringle near the lysine ligand binding site. Selected segments of the LDL apo B-100 sequence that contain free sulfhydryl cysteines were subjected to energy minimization and docking with the ligand binding site and adjacent regions of the LPaK9 model. In the docking experiments, apo B-100 segment 3732-3745 (PSCKLDFREIQIYK) displayed the best fit and the largest number of van der Waals contacts with models of LPaK9. Other apo B-100 peptides with sulfhydryl cysteine were found to be less compatible when minimized with this kringle. These results support and extend previously suggested mechanisms for a complex interaction between apo[a] and apo B-100 that involve more than a simple covalent disulfide bond.     

ATP‐independent molecular chaperone activity generated under reducing conditions

Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra‐ to intermolecular disulfide bond relay mechanism. Chaperone‐active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol‐containing compounds and proteins, and appear in parallel with reduction‐induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space.SignificanceChaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction‐induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.     

Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125

Streptococcus pyogenes and other Gram‐positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild‐type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125^Cys426Ala. We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands. Proteins 2014; 82:517–527. © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

The multisubunit structure of synaptophysin. Relationship between disulfide bonding and homo-oligomerization

Synaptophysin, a major membrane protein of synaptic vesicles, contains four transmembrane regions and two intravesicular loops. Synaptophysin monomers associate into homopolymers that have the potential to form channels in the synaptic vesicle membrane. Here we show that in native synaptophysin, homopolymers are linked by noncovalent forces. The molecule contains unstable intramolecular disulfide bonds that undergo disulfide exchange during solubilization, thereby covalently cross-linking neighboring synaptophysin molecules. The locations of the intramolecular disulfide bonds in synaptophysin were determined, revealing that each of the two intravesicular loops of synaptophysin is circularized by a single disulfide bond. Cross-linking of synaptophysin by disulfide bonds can be triggered in synaptic vesicles and in intact cells by a cycle of reduction and oxidation, suggesting that native synaptophysin is a homomultimer in situ. In addition, chemical cross-linking of native synaptophysin demonstrates that a low molecular weight protein is specifically associated with synaptophysin complexes and is lost upon reduction of the intramolecular disulfide bonds. These data suggest that native synaptophysin forms a noncovalent homomultimeric complex whose structure and interaction with other proteins are dependent on the integrity of its intramolecular disulfide bonds and phospholipid environment.     

Identification of thiol groups and a disulfide crosslink site in bovine myelin proteolipid protein

The existence of disulfide crosslinks limits the number of possible folded structures a protein can assume. Thus localization of disulfide and thiol groups is a key to understanding the conformation and orientation of myelin proteolipid protein (PLP) in the myelin membrane. [¹⁴C]Carboxamidomethylated PLP was fragmented with chymotrypsin, and the resulting mixture was partially separated by reversed-phase HPLC. Purified ¹⁴C-labeled peptides and a disulfide containing peptide were characterized by amino acid analysis. These experiments showed that Cys-32 and Cys-34 are free thiols, and are presumably on the interior of the cell or within the membrane bilayer, and that Cys-200 and Cys-219 are joined by a disulfide bond, and are probably located on the extracellular face of the membrane. Sequence analysis experiments indicate that Cys-5, Cys-6 and Cys-9 are linked by disulfides, probably to other parts of the protein on the extracellular face of the membrane.     

The role of Dsb proteins of Gram-negative bacteria in the process of pathogenesis

Tertiary and quaternary structures of extracytoplasmic proteins containing more than one cysteine residue often require introduction of disulfide bonds. This process takes place in an oxidative environment, such as the periplasm of Gram-negative bacteria, and is catalyzed by Dsb (disulfide bond formation) proteins. Mutations in dsb genes influence the conformation and stability of many extracytoplasmic proteins. Thus, many pathogens become partially or fully attenuated due to improper folding of proteins that act as virulence factors. This review summarizes the current knowledge on Dsb proteins and their effect on the pathogenicity of Gram-negative bacteria. The potential application of Dsb proteins in biotechnology is also discussed.     

Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles

Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.