Disulfide Loop Research Articles

Stabilization of HIV-1 gp120-CD4 Receptor Complex through Targeted Interchain Disulfide Exchange

HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser(60) on the CD4 domain 1 alpha-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys(126)-Cys(196)), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys(60) and gp120 Cys(126), and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied.

Redox Regulation of Transglutaminase 2 Activity

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys(230), Cys(370), and Cys(371) that is involved in oxidative inactivation of TG2. Within this triad, Cys(370) was found to participate in disulfide bonds with both Cys(230) and its neighbor, Cys(371). Notably, Ca(2+) was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys(230) appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys(370)-Cys(371) through formation of the Cys(230)-Cys(370) disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys(230) is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.

A Low-Molecular-Weight Entry Inhibitor of both CCR5- and CXCR4-Tropic Strains of Human Immunodeficiency Virus Type 1 Targets a Novel Site on gp41

A low-molecular-weight human immunodeficiency virus type 1 (HIV-1) inhibitor, PF-68742 (molecular weight, 573), has been identified in a high-throughput screen for compounds that block HIV-1 envelope glycoprotein (Env)-mediated fusion. The compound is shown to be potent against R5 and X4 isolates in both cell-cell fusion and antiviral assays (50% effective concentrations of approximately 0.1 to 1 muM). Postfusion and HIV-1 pseudotyping control experiments confirm that PF-68742 is an entry inhibitor with Env as the specific target for antiviral action. PF-68742 was not able to block binding of monomeric gp120 to soluble CD4 or the binding of gp120:CD4 complexes to cell-associated CCR5, thus distinguishing PF-68742 from described gp120 antagonists and coreceptor binders. Escape variants of HIV-1(NL4-3) were selected, and all resistant viruses were found to contain a common G514R (HxB2 numbering) mutation in Env, located proximal to the furin cleavage site in the fusion peptide of gp41. When introduced into wild-type NL4-3 gp41, G514R conferred resistance to PF-68742. Resistance via G514R is shown to be associated with enhancement of virion infectivity by PF-68742 that may result from altered properties of inhibitor-bound Env, rather than from a loss of compound binding. Wild-type viruses and those with substitutions in the disulfide loop (DSL) region of gp41 were also examined for PF-68742 sensitivity. Here, complete resistance to PF-68742 was found to occur through changes outside of position 514, including in the gp41 DSL region. The results highlight PF-68742 as a starting point for novel therapies against HIV-1 and provide new insights into models of Env-mediated fusion.

New insights into the biological activity and secretion properties of a polypeptide derived from tilapia somatotropin

In a previous study, we unexpectedly found that tilapia growth hormone (tiGH) secreted to the culture media by transformed cells of the yeast Pichia pastoris lacks 46 amino acids from the C-terminal end. In the present study, we cloned the exact fragment that code for this truncated variant and demonstrated its growth promoting activity in goldfish when it's administered by immersion bath. Furthermore, a better characterization of this polypeptide was performed. Administration of the polypeptide derived from tiGH increased superoxide anion production and has a mitogenic effect on peripheral blood leukocytes. This molecule binds to liver membranes proteins in vitro in a saturable manner. Beside, we cloned and expressed tiGH and its truncated variant in mammalian cells using the signal peptide of this hormone and we observed that the secretion was drastically reduced in the truncated tiGH as compared to the intact molecule. Truncated tilapia growth hormone lacking the helix 4 and two disulfide loops is still a bioactive hormone, suggesting that the disulfide bonds and the helix 4 are not essential for the biological activities examined in this work. However, the growth hormone C-terminal portion seems to be essential for this hormone to be secreted by cultured cells in vitro.

Evidence for a Partially Structured State of the Amylin Monomer

Islet amyloid polypeptide (amylin) is the main component in amyloid deposits formed in type II diabetes. We used triplet quenching to probe the dynamics of contact formation between the N-terminal disulfide loop and a C-terminal tryptophan in monomeric amylins from human and rat. Quenching rates measured in the absence of denaturant are four times larger than those in 6 M guanidinium chloride, indicating a decrease in the average end-to-end distance (collapse) at low denaturant concentrations. We were surprised to find an even greater (sevenfold) increase in quenching rates on removal of denaturant for a hydrophilic control peptide containing the disulfide loop compared to the same peptide without the loop (twofold change). These results suggest that collapse is driven by backbone-backbone and backbone-side chain interactions involving the disulfide loop portion of the chain rather than by the formation of side-chain hydrophobic contacts. Molecular dynamics simulations of the control peptide show that the collapse results from hydrogen-bonding interactions between the central residues of the chain and the disulfide loop. The quenching experiments also indicate that the monomer of the human, amyloidogenic form of amylin is more compact than the rat form, which does not form amyloid. We discuss these newly observed differences between human and rat amylin in solution and their possible relation to aggregation and to the physiological function of amylin binding to the calcitonin receptor.

The Role of the VWF A1 and A2 Domains in Murine Models of TTP and Thrombosis.

Abstract 3060Poster Board II-1036von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein synthesized in endothelial cells and megakaryocytes. In humans and mice, VWF dysfunction is associated both with defects in hemostasis, and with the systemic blood clotting disease thrombotic thrombocytopenic purpura (TTP). The initial adhesion of platelets to sites of vascular injury in large part involves binding of the VWF A1 domain to the platelet glycoprotein receptor GPIb alpha. This VWF A1-GPIb alpha interaction, along with deficiency of the ADAMTS13 plasma metalloprotease, is thought to be required for the pathogenesis of TTP. Deficiency of ADAMTS13 results in the failure to cleave the Y1605-M1606 sissile bond within the A2 domain of VWF. The structure of VWF is strongly influenced by its high content of cysteine residues, all of which are involved in inter-or intra-chain disulfide bonds. The location of these cysteine residues within the A domains leads to the formation of disulfide loops within the A1 (Cys 1272-1458) and A3 (Cys 1686-1872) domains, but not within the A2 domain. The lack of a disulfide loop allows the A2 domain to assume a “flexible” conformation that is thought to “open” in response to fluid shear stress, exposing the Y1605-M1606 bond to cleavage by ADAMTS13. To investigate specifically the role of the VWF A1-Gp1b alpha interaction in the context of otherwise functional VWF in vivo, we generated a chimeric murine VWF expression construct in which the murine A1 domain sequence is replaced with the corresponding sequence from human VWF (the human VWF A1 domain is known to not interact appreciably with murine GPIb alpha). Additionally, we engineered a VWF construct in which paired cysteine residues analogous to those in the A1 and A3 domains were introduced into the A2 domain sequence, with the goal being to “lock” the A2 domain closed and prevent cleavage by ADAMTS13. Hydrodynamic tail vein injection of both the VWF-hA1 and the VWF-A2 lock constructs into VWF-deficient mice resulted in plasma VWF levels up to 20-fold higher than observed in wild-type mice, dependent on the amount of plasmid injected. Importantly, the degree of VWF multimerization appeared nearly identical both to that observed in wild-type mice, and to mice injected with wild-type murine VWF, and expression persisted for approximately 30 days. Functionally, unlike WT murine VWF, expression of VWF-hA1 failed to restore thrombus formation in a ferric chloride-induced injury model, demonstrating the crucial importance of the VWF A1-GP1b apha interaction in thrombus formation. Currently we are investigating whether expression of VWF-hA1 can support disease pathogenesis in a mouse model of TTP. Similarly, we are determining whether expression of VWF-A2 lock leads to development of TTP, even in the presence of ADAMTS13. The ultimate goal of these studies is to completely “humanize” the VWF A1-GP1b alpha interaction in mice by replacing the murine GP1b alpha sequence with that from humans. These resulting animals will be used to further investigate the role of the VWF A1 domain-GPIb alpha interaction in vivo, and should prove useful for identifying compounds to effectively inhibit this interaction in humans. In addition, the expression of a VWF construct that is unable to be cleaved by ADAMTS13 should help to elucidate the role of VWF cleavage in TTP pathogenesis. DisclosuresNo relevant conflicts of interest to declare.

Research retrospect on small molecule protease in-hibitors and peptide toxins

This paper is a retrospect of our research works on small molecular weight protease inhibitors and peptide toxins, all of them are active peptides. The Lysin active fragment of the double headed mung bean trypsin inhibitor has been intensively studied. The approaches of sequence determination, gene expression and mutagenesis as well as peptide synthesis are used to indicate that the essen- tial core of the inhibitor is composed of two conjugated disulfide loops each with nine-residues, which shares high homology with the cyclic peptide sun flower trypsin inhibitor. Another described inhibitor is the Trichosanthes trypsin inhibitor belonging to the squish family, its gene sequence was first reported in this family. The studied peptide toxins include scorpion toxins and conotoxins. Many novel potassium channel toxins were found from the scorpion Buthus martensi Karsch, in- cluding the calcium dependent BK or SK potassium channel toxins and voltage dependent toxins. Some of them have two deferent functions displaying at separated region, their structure-function relationships were elucidated. The expressed recombinant scorpion chloride toxin has a potential in treatment of malignant gliomas. From 16 species of cone snail 50 novel conotoxin peptides were identified, and 134 novel conotoxin genes were cloned. Three unreported superfamilies were characterized, designated as V, Y, K superfamily, respectively; some new disulfide frame and arrangement, unique motif, D-form residue in the toxin primary structures were found; the genomic structure of A superfamily was analysed, in the intro there is a very conserved sequence at the 3’end which could be used as a primer to clone more new conotoxin genes; the biological function of some conotoxins were elucidated.

Peptide bond scission of staphylococcal enterotoxin C2 and related factors

To investigate the limited digestion of recombinant staphylococcal enterotoxin C2 (SEC2-His)in different conditions. The purified recombinant SEC2-His was treated with different reagents and the cleavage of rSEC2 molecule was observed by SDS-PAGE. The cleavage occurred in positions Cys93-Cys110 of the disulfide loop. Complete auto-cleavage of recombinant SEC2 was observed in solution at 37degrees within 24 hrs, and that was accelerated under alkaline conditions. The auto-cleavage of the recombinant protein was inhibited in the presence of beta-ME (2%), PMSF (5-10 mmol/L), imidazole (1 mol/L) or crude E.coli lysate. Non-specific degradation of recombinant SEC2 was promoted with the increasing of the concentration of H(2)O(2). The recombinant SEC2-His is broken down in special site of protein, which may be associated with the protein structure.

Advantages of a Synthetic Peptide Immunogen Over a Protein Immunogen in the Development of an Anti‐Pilus Vaccine for Pseudomonas aeruginosa

The type IV pilus is an important adhesin in the establishment of infection by Pseudomonas aeruginosa. We have previously reported on a synthetic peptide vaccine targeting the receptor-binding domain of the main structural subunit of the pilus, PilA. The receptor-binding domain is a 14-residue disulfide loop at the C-terminal end of the pilin protein. The objective of this study was to compare the immunogenicity of a peptide-conjugate to a protein subunit immunogen to determine which was superior for use in an anti-pilus vaccine. BALB/c mice were immunized with the native PAK strain pilin protein and a synthetic peptide of the receptor-binding domain conjugated to keyhole limpet haemocyanin. A novel pilin protein with a scrambled receptor-binding domain was used to characterize receptor-binding domain-specific antibodies. The titres against the native pilin of the animals immunized with the synthetic peptide-conjugate were higher than the titres of animals immunized with the pilin protein. In addition, the affinities of anti-peptide sera for the intact pilin receptor-binding domain were significantly higher than affinities of anti-pilin protein sera. These results have significant implications for vaccine design and show that there are significant advantages in using a synthetic peptide-conjugate over a subunit pilin protein for an anti-pilus vaccine.

Molecular Basis of the Interactions of the Nogo-66 Receptor and Its Homolog NgR2 with Myelin-Associated Glycoprotein: Development of NgROMNI-Fc, a Novel Antagonist of CNS Myelin Inhibition

Myelin-associated glycoprotein (MAG) is a sialic acid-binding Ig-family lectin that functions in neuronal growth inhibition and stabilization of axon-glia interactions. The ectodomain of MAG is comprised of five Ig-like domains and uses neuronal cell-type-specific mechanisms to signal growth inhibition. We show that the first three Ig-like domains of MAG bind with high affinity and in a sialic acid-dependent manner to the Nogo-66 receptor-1 (NgR1) and its homolog NgR2. Domains Ig3-Ig5 of MAG are sufficient to inhibit neurite outgrowth but fail to associate with NgR1 or NgR2. Nogo receptors are sialoglycoproteins comprised of 8.5 canonical leucine-rich repeats (LRR) flanked by LRR N-terminal (NT) and C-terminal (CT)-cap domains. The LRR cluster is connected through a stalk region to a membrane lipid anchor. The CT-cap domain and stalk region of NgR2, but not NgR1, are sufficient for MAG binding, and when expressed in neurons, exhibit constitutive growth inhibitory activity. The LRR cluster of NgR1 supports binding of Nogo-66, OMgp, and MAG. Deletion of disulfide loop Cys(309)-Cys(336) of NgR1 selectively increases its affinity for Nogo-66 and OMgp. A chimeric Nogo receptor variant (NgR(OMNI)) in which Cys(309)-Cys(336) is deleted and followed by a 13 aa MAG-binding motif of the NgR2 stalk, shows superior binding of OMgp, Nogo-66, and MAG compared with wild-type NgR1 or NgR2. Soluble NgR(OMNI) (NgR(OMNI)-Fc) binds strongly to membrane-bound inhibitors and promotes neurite outgrowth on both MAG and CNS myelin substrates. Thus, NgR(OMNI)-Fc may offer therapeutic opportunities following nervous system injury or disease where myelin inhibits neuronal regeneration.

Collapse Of Rat And Human Amylin From Nanosecond-resolved Intramolecular Contact Formation

Amylin is a 37 residue peptide with hormone properties related to nutrient intake regulating glucose levels. It is found in the form of amyloid deposits in the β-cells of type II diabetic patients. Similar to α-syn and Aβ, it is an intrinsically disordered protein. Little is known about amylin's conformational properties in solution and their relation to function and aggregation.We have used triplet quenching to monitor the dynamics of end-to-end contact formation between the N-terminal disulfide loop of human and rat amylin and a C-terminal tryptophan. The quenching rates for both species increase significantly in aqueous buffer relative to 6M guanidinium chloride (GdmCl), indicating a decrease in the average end-to-end distance. Comparisons with control peptides suggest that backbone-backbone interactions, involving the N-terminal disulfide loop are the principal driving force for collapse in these peptides, rather than sidechain-sidechain hydrophobic interactions. Molecular dynamics simulations on the control sequences indicate that the collapse results from hydrogen-bonding interactions between the central residues of the chain and the disulfide loop, reducing the length of the free chain by ∼ 2-fold. This structural feature may contribute to the functional role of the disulfide loop in amylin and in the larger family of calcitonin gene-related peptides. We discuss the newly observed differences between monomeric human and rat IAPP in solution and their possible relation to aggregation.

The Cotranslational Maturation Program for the Type II Membrane Glycoprotein Influenza Neuraminidase

The earliest steps in nascent protein maturation greatly affect its overall efficiency. Constraints placed on maturing proteins at these early stages limit available conformations and help to direct the native maturation process. For type II membrane proteins, these cotranslational constraints include N- and C-terminal membrane tethering, chaperone binding, and disulfide bond formation. The cotranslational maturation process for the type II membrane glycoprotein influenza neuraminidase (NA) was investigated to provide a deeper understanding of these initial endoplasmic reticulum events. The type II orientation provides experimental advantages to monitor the first maturation steps. Calnexin was shown to cotranslationally interact with NA prior to calreticulin. These interactions were required for the efficient maturation of NA as it prematurely formed intramolecular disulfides and aggregated when calnexin and calreticulin interactions were abolished. Lectin chaperone binding slowed the NA maturation process, increasing its fidelity. Carbohydrates were required for NA maturation in a regio-specific manner. A subset of NA formed intermolecular disulfides and oligomerized cotranslationally. This fraction increased in the absence of calnexin and calreticulin binding. NA dimerization also occurred for an NA mutant lacking the critical large loop disulfide bond, indicating that dimerization did not require proper NA oxidation. The strict evaluation of proper maturation carried out by the quality control machinery was instilled at the tetramerization step. This study illustrates the type II membrane protein maturation process and shows how important cotranslational events contribute to the proper cellular maturation of glycoproteins.

Incremental Improvement in Bioengineering of Coagulation Factor VIII for Efficient Expression: Elimination of a Dispensable Disulfide Loop Enhances Secretion

Coagulation factor VIII (FVIII), a glycoprotein cofactor, performs a critical function in the intrinsic blood coagulation pathway and a quantitative or qualitative deficiency of this protein results in Hemophilia A. The primary obstacle to producing low cost replacement therapy is the inherent limitation of commercial recombinant FVIII (rFVIII) production since its expression is 2 to 3 orders of magnitude lower than that of other comparably sized proteins. Inefficient mRNA expression, protein misfolding & chaperone mediated retention and the requirement for facilitated transport from the Endoplasmic Reticulum (ER) to Golgi have been identified as the major bottlenecks to efficient expression of rFVIII. Bioengineering strategies aimed at clearing each of these hurdles have resulted in the generation of several rFVIII variants with more efficient expression. This study focused on the effect of each of FVIII's eight disulfide bonds on its secretion efficiency and functionality. Elimination of specific disulfide loops has previously been shown to confer a secretion advantage in certain proteins, e.g. lysozyme and human chorionic gonadotropin (hCG). The FVIII disulfide bonds were eliminated by replacing the corresponding cysteine residues with glycine residues by a PCR-based site-directed mutagenesis protocol using mutagenic primers. The disulfide mutants were created both in the full length FVIII as well as a previously bioengineered FVIII variant (226/N6) with enhanced (5–10 fold) secretion efficiency. All the mutants were characterized by restriction enzyme digestion and DNA sequencing. The secretion efficiency of all the mutants was studied by transient transfection in Chinese Hamster Ovary (CHO) and COS-1 cells. FVIII activity was measured by one-stage and two-stage clotting assays and the antigen levels were quantified by anti-FVIII light chain sandwich enzyme-linked immunosorbent assay. Seven of the eight disulfide bonds in factor VIII were found to be indispensable for the correct folding and secretion since all these disulfide mutants were retained intracellularly. A single mutant with substitution of glycine for cysteine at position 2326 in the C2 domain resulted in secretion of a functionally inactive protein. Elimination of the disulfide loop between cysteine residues at positions 1899 and 1903 in the A3 domain resulted in a 1.5- to 2-fold increase in secretion of both the full length FVIII and the already efficiently expressed 226/N6 rFVIII variant. This enhanced secretion was reflected in both the antigen levels and the activity of the protein. Also, incorporation of these mutations in 226/N6 F309S, yet another efficiently secreted rFVIII variant, further improved secretion with a synergistic effect, resulting overall in a 25- to 30-fold higher expression than full length FVIII in CHO and COS-1 cells. Interestingly, coagulation factor V (FV), that shares considerable homology with FVIII and is secreted much more efficiently, lacks a disulfide loop in the homologous position to the 1899–1903 bridge with FVIII. The other seven disulfide bonds are conserved between these two proteins. Such combined targeted bioengineering strategies may facilitate more efficient production of rFVIII toward lower cost replacement therapy.

Production of metal ion-dependent monoclonal antibodies against peptides in bovine prothrombin fragment 1

Bovine prothrombin fragment 1 (F-1: the amino-terminal 156 residues of prothrombin) is used as a model to study the Ca(II) and phospholipid binding of prothrombin. The 35-46 segment in F-1 posses an alpha-helical region and three aromatic residues, conserved in several vitamin K-dependent blood coagulation factors. These residues are believed to have a specific function and to be important in the phospholipid binding of F-1. The 47-62 region, a disulfide loop, is believed to stabilize the gamma-carboxyglutamic acid domain of the protein. Goals of this research were to produce monoclonal antibodies against the above two sequences, for later functional studies. Antibodies S9-32.8 and S9-5.5 were produced against the 35-46 sequence; antibody S11-23.4 was raised against the 47-62 region. Both S9-32.8 and S9-5.5 bound to F-1 immobilized on ELISA plates in the presence of 10 mM Ca(II) with higher affinity than to F-1 coated in the presence of 10 mM Mg(II) or in the absence of metal ions. S11-23.4 showed greatest binding to F-1 coated in the presence of 10 mM Mg(II). Thus, the epitopes of the antibodies are metal ion-dependent and are developed by Ca(II) binding to F-1.

Role of the HIV gp120 Conserved Domain 1 in Processing and Viral Entry

The importance of the N-terminal region of HIV gp120 conserved domain 1 (gp120-C1) to envelope function has been examined by alanine-scanning mutagenesis and subsequent characterization of the mutagenic effects on viral entry; envelope expression, processing, and incorporation; and gp120 association with gp41. With respect to the wild-type gp120, mutational effects on viral entry fall into two classes: functional, as defined by >20% entry with respect to wild type, and impaired, as defined by <20% entry with respect to wild type. Based on Western blot analyses of cell lysates and virions, the entry impairment of W35A, V38A, Y39A, Y40A, G41A, V42A, and I52A is due primarily to disruption of envelope processing. The entry impairment of P43A and W45A is apparently due to a combination of effects on processing and incorporation into virions. In contrast, the entry impairment of V44A and F53A is primarily due to disruption of the gp120-gp41 interaction, which results in dissociation of gp120 from the virion. We present a model for gp120-C1 interactions with gp120-C5 and the gp41 disulfide loop in unprocessed gp160 and processed gp120/gp41.

Factors Governing Electron Capture by Small Disulfide Loops in Two-Cysteine Peptides

Integrated molecular orbital-molecular orbital (IMOMO) calculations on 17 short disulfide-bridged peptides (up to 16 residues, with at most five intraloop residues) were performed to elucidate some factors controlling their electron capture. These illustrative systems display contrasted behaviors, shedding light on several criteria of differentiation: size, shape, and rigidity of the disulfide-linking loop, intramolecular hydrogen bonds, etc. The geometrical malleability of disulfide radical anions, whose existence and role as intermediate have been evidenced, is discussed. The disulfide elongation (by ca. 0.7 A) upon electron capture induces "soft" structural damages for these turn structures, with a weakening or cleavage of vicinal hydrogen bond(s). On the basis of a series of six Cys-Alan-Cys peptides, it is proposed that electron affinity reflects the topological frustration of these short and highly constrained structures. Results for a series of amino acid mutations are analyzed for the Cys-Xxx-Yyy-Cys motif, common to redox enzymes of the thioredoxin superfamily.

Oxidative Folding and Assembly with Transthyretin Are Sequential Events in the Biogenesis of Retinol Binding Protein in the Endoplasmic Reticulum

Retinol-binding protein (RBP) is secreted out of the cell in its ligand-bound holo-form. The apo-form of RBP is selectively retained within the endoplasmic reticulum (ER) by a mechanism that remains unknown. Using isolated microsomal system, we have recapitulated the biogenesis of RBP involving its oxidative folding and assembly with transthyretin in the ER. In addition to dissecting its pathway of disulfide oxidation, we have analyzed association of its early folding intermediates with ER-chaperones. Our results show that of the three intramolecular disulfides present in RBP (4-160, 70-174, and 120-129) the smallest loop (120-129) was most critical for RBP to fold. Its absence caused RBP to aggregate into an intermolecular disulfide-linked structure. After acquisition of the small loop, formation of one of the two big disulfides (4-160 or 70-174) was sufficient for RBP to acquire a folded state. Using cross-linking in intact microsomes and sedimentation on sucrose gradients, we show that newly synthesized RBP is associated with a complex of chaperones consisting of Grp94, BiP, PDI, and calnexin. The complex was constitutively present in the ER, independent of the presence of folding substrates. RBP dissociated from this complex coincident with the formation of one of the two big disulfide loops, whereas RBP mutant lacking both the large disulfides showed persistent association. While highlighting the matrix-like characteristics of ER in isolated microsomal system our results provide insight into RBP folding and assembly mechanisms that will aid our understanding of its complex secretion properties.

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.

Catalytic Cycle of Human Glutathione Reductase Near 1 Å Resolution

Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 Å that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity—a net deviation of 53° across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.

Conantokin-P, an unusual conantokin with a long disulfide loop

The conantokins are a family of Conus venom peptides (17–27AA) that are N-methyl- d-aspartate (NMDA) receptor antagonists. Conantokins lack disulfide bridges (six out of seven previously characterized peptides are linear), but contain multiple residues of γ-carboxyglutamate. These post-translationally modified amino acids confer the largely helical structure of conantokins by coordinating divalent metal ions. Here, we report that a group of fish-hunting cone snails, Conus purpurascens and Conus ermineus, express a distinctive branch of the conantokin family in their venom ducts. Two novel conantokins, conantokin-P (Con-P) and conantokin-E (Con-E) are 24AA long and contain five γ-carboxyglutamate residues. These two peptides are characterized by a long disulfide loop (12 amino acids including two Gla residues between the Cys residues). The oxidative folding studies of Con-P revealed that the formation of the disulfide bond proceeded significantly faster in the presence of Ca ++ ions. Circular dichroism suggested that Con-P is less helical than other previously characterized conantokins. Con-P blocks NMDA receptors containing NR2B subunit with submicromolar potency. Furthermore, the subtype-selectivity for different NR2 subunits differs from that of the previously characterized conantokins. Our results suggest that different branches of the phylogenetic tree of cone snails have evolved distinct groups of conantokins, each with its own unique biochemical features.