Importance of conserved cysteine residues in the coronavirus envelope protein

Coronavirus envelope (E) proteins play an important, not fully understood role(s) in the virus life cycle. All E proteins have conserved cysteine residues located on the carboxy side of the long hydrophobic domain, suggesting functional significance. In this study, we confirmed that mouse hepatitis coronavirus A59 E protein is palmitoylated. To understand the role of the conserved residues and the necessity of palmitoylation, three cysteines at positions 40, 44, and 47 were changed singly and in various combinations to alanine. Double- and triple-mutant E proteins resulted in decreased virus-like particle output when coexpressed with the membrane (M) protein. Mutant E proteins were also studied in the context of a full-length infectious clone. Single-substitution viruses exhibited growth characteristics virtually identical to those of the wild-type virus, while the double-substitution mutations gave rise to viruses with less robust growth phenotypes indicated by smaller plaques and decreased virus yields. In contrast, replacement of all three cysteines resulted in crippled virus with significantly reduced yields. Triple-mutant viruses did not exhibit impairment in entry. Mutant E proteins localized properly in infected cells. A comparison of intracellular and extracellular virus yields suggested that release is only slightly impaired. E protein lacking all three cysteines exhibited an increased rate of degradation compared to that of the wild-type protein, suggesting that palmitoylation is important for the stability of the protein. Altogether, the results indicate that the conserved cysteines and presumably palmitoylation are functionally important for virus production.     

Roles of two conserved cysteine residues in the activation of human adenovirus proteinase.

The roles of two conserved cysteine residues involved in the activation of the adenovirus proteinase (AVP) were investigated. AVP requires two cofactors for maximal activity, the 11-amino acid peptide pVIc (GVQSLKRRRCF) and the viral DNA. In the AVP-pVIc crystal structure, conserved Cys104 of AVP has formed a disulfide bond with conserved Cys10 of pVIc. In this work, pVIc formed a homodimer via disulfide bond formation with a second-order rate constant of 0.12 M(-1) s(-1), and half of the homodimer could covalently bind to AVP via thiol-disulfide exchange. Alternatively, monomeric pVIc could form a disulfide bond with AVP via oxidation. Regardless of the mechanism by which AVP becomes covalently bound to pVIc, the kinetic constants for substrate hydrolysis were the same. The equilibrium dissociation constant, K(d), for the reversible binding of pVIc to AVP was 4.4 microM. The K(d) for the binding of the mutant C10A-pVIc was at least 100-fold higher. Surprisingly, the K(d) for the binding of the C10A-pVIc mutant to AVP decreased at least 60-fold, to 6.93 microM, in the presence of 12mer ssDNA. Furthermore, once the mutant C10A-pVIc was bound to an AVP-DNA complex, the macroscopic kinetic constants for substrate hydrolysis were the same as those exhibited by wild-type pVIc. Although the cysteine in pVIc is important in the binding of pVIc to AVP, formation of a disulfide bond between pVIc and AVP was not required for maximal stimulation of enzyme activity by pVIc.     

The function of conserved cysteine residues in the extracellular domain of human receptor-activity-modifying protein

The receptor-activity-modifying protein (RAMP) 1 is a single-transmembrane-domain protein associated with the calcitonin-like receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys(27) in myc-tagged human (h) RAMP1 was deleted (hRAMP1Delta1), and Cys(40), Cys(57), Cys(72), Cys(82) and Cys(104) were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Delta1 or -C82A, cell surface expression, [(125)I]halphaCGRP binding and cAMP formation in response to halphaCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24+/-7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [(125)I]halphaCGRP binding of hCLR/myc-hRAMP1-C72A was 13+/-3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by halphaCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14+/-1% and 33+/-2% of that of the hCLR/myc-hRAMP1 with comparable EC(50). But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP receptor function was not affected by the deletion of Cys(27) or the substitution of Cys(82) by Ala in hRAMP1, but it was impaired by the substitution of Cys(40), Cys(57), Cys(72) and Cys(104) by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

Proteolytic processing of the Ada protein that repairs DNA O6-methylguanine residues in E. coli

In extracts of E. coli treated with an adapting regime of MNNG, the induced 39kd Ada protein having O6-MeG-DNA methyltransferase activity is processed to a 19kd active domain corresponding to the C-terminal half of the intact protein. This proteolytic processing has been followed on Western immunoblots using antisera raised against the 19kd fragment. Initial processing at 25 degrees C or 37 degrees C mainly generates a fragment of mol. wt. 24kd which then undergoes a slower second cleavage to generate the 19kd active domain. Preceding this second cleavage site is a sequence of amino acids Thr- -Gly-Met-Thr- -Lys that also occurs at another site in the N-terminal half of the 39kd methyltransferase. It is proposed that this sequence is a recognition site for proteolytic activity. On the basis of cleavage of the Ada protein at either one or both of these sites, fragments may be generated of mol. wt. 24kd and 19kd containing the active site for O6-methylguanine and O4-methylthymine repair, and 15kd and 20kd, containing the active site for methylphosphotriester repair. These observations explain previous reports by others on the existence in cell extracts of multiple methyltransferase activities of different sizes recognizing O-methyl lesions in DNA. The cellular protease involved is resistant to a wide range of protease inhibitors.     

Export of the periplasmic maltose-binding protein ofEscherichia coli

The export of the maltose-binding protein (MBP), themalE gene product, to the periplasm ofEschericha coli cells has been extensively investigated. The isolation of strains synthesizing MalE-LacZ hybrid proteins led to a novel genetic selection for mutants that accumulate export-defective precursor MBP (preMBP) in the cytoplasm. The export defects were subsequently shown to result from alterations in the MBP signal peptide. Analysis of these and a variety of mutants obtained in other ways has provided considerable insight into the requirements for an optimally functional MBP signal peptide. This structure has been shown to have multiple roles in the export process, including promoting entry of preMBP into the export pathway and initiating MBP translocation across the cytoplasmic membrane. The latter has been shown to be a late event relative to synthesis and can occur entirely posttranslationally, even many minutes after the completion of synthesis. Translocation requires that the MBP polypeptide exist in an export-competent conformation that most likely represents an unfolded state that is not inhibitory to membrane transit. The signal peptide contributes to the export competence of preMBP by slowing the rate at which the attached mature moiety folds. In addition, preMBP folding is thought to be further retarded by the binding of a cytoplasmic protein, SecB, to the mature moiety of nascent preMBP. In cells lacking this antifolding factor, MBP export represents a race between delivery of newly synthesized, export-competent preMBP to the translocation machinery in the cytoplasmic membrane and folding of preMBP into an export-incompetent conformation. SecB is one of threeE. coli proteins classified as molecular chaperones by their ability to stabilize precursor proteins for membrane translocation.     

Site directed mutagenesis of two cysteine residues in the E. coli ogt O6-alkylguanine DNA alkyltransferase protein.

The E. coli ogt O6-alkylguanine-DNA alkyltransferase has two cysteine residues positioned identically with respect to cysteines in the E. coli ada O6-alkylguanine-DNA alkyltransferase. In order to assess their function, these residues were each substituted by a glycine to generate altered forms of the ogt protein. Mutagenesis of cysteine-139, located within a 'PCHRV' region of homology, eliminated functional activity confirming that this residue is the methyl-accepting cysteine in the active site of the protein. Substitution of cysteine 102 within the sequence 'LRTIPCG' had little effect on the ogt protein activity demonstrating that this cysteine is not directly involved with the transfer of O6-methylguanine adducts.     

Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein

Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2′-dGTP (8-oxo-dGTP) to the monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA. Bacterial and mammalian homologs of MutT protein share the phosphohydrolase module (MutT: Gly37→Gly59). By saturation mutagenesis of conserved residues in the MutT module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were revealed to be essential to suppress spontaneous A:T→C:G transversion mutation in a mutT^– mutator strain. For the other six residues (Lys39, Glu44, Thr45, Arg52, Glu56 and Gly59), many positive mutants which can suppress the spontaneous mutation were obtained; however, all of the positive mutants for Glu44 and Arg52 either partially or inefficiently suppressed the mutation, indicating that these two residues are also important for MutT function. Several positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the elevated spontaneous mutation rate, as seen with the wild-type, hence, these four residues are non-essential for MutT function. As Lys38 and Glu55 in human MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could not be replaced by any other residue without loss of function, different structural features between the two modules of MTH1 and MutT proteins are evident.     

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.     

Analysis of the role of conserved cysteine residues in the bcl-2 oncoprotein

The Bcl-2 oncoprotein is an integral membrane protein localized primarily to the outer membrane of the mitochondria. The precise molecular mechanism responsible for the antiapoptotic action of Bcl-2 remains unknown. Two cysteine residues are found in Bcl-2 and these residues are well-conserved across species. The first cysteine (cys(155)) is located in the alpha5 domain, a region important for the ion channel properties of Bcl-2, while the second cysteine (cys(226)) is located in the carboxyl-terminal membrane anchor domain. In this study, we found that replacement of both cysteines with serine residues generated a mutant protein that retained the ability to homodimerize and heterodimerize with proapoptotic Bax protein in vitro. In whole cells, the mutant protein efficiently heterodimerized with Bax, but exhibited impaired homodimerizationrelative to wild-type Bcl-2. The mutant protein was also less efficient than wild-type Bcl-2 at suppressing caspase activation, DNA fragmentation, and loss of viability during IL-3 withdrawal-induced apoptosis. Together, the data indicate that the cysteine residues in Bcl-2 contribute, but are not absolutely essential, to the ability of Bcl-2 to homodimerize, heterodimerize with Bax, and suppress apoptosis.     

Processing of Ada protein by two serine endoproteases Do and So from Escherichia coli

Two soluble serine proteases Do and So from Escherichia coli were found to distinctively cleave the purified, 39 kDa Ada protein into fragments with sizes of 12-31 kDa. Protease So appears to generate a C-terminal 19 kDa polypeptide, similarly to OmpT protease. In addition, the purified 19 kDa C-terminal half of Ada protein can be further processed mainly to an 18 kDa fragment by protease So and to a 12 kDa by protease Do. These results suggest that proteases Do and So are involved in endogenous cleavage of Ada protein, which may play a role in down-regulating the adaptive response to alkylating agents.     

The role of conserved residues of chagasin in the inhibition of cysteine peptidases

We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10-100-fold increases in the K(i) for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.     

Reactivity of the two essential cysteine residues of the periplasmic mercuric ion-binding protein, MerP

Reactivities of the two essential cysteine residues in the heavy metal binding motif, MTC(14)AAC(17), of the periplasmic Hg(2+)-binding protein, MerP, have been examined. While Cys-14 and Cys-17 have previously been shown to be Hg(2+)-binding residues, MerP is readily isolated in an inactive Cys-14-Cys-17 disulfide form. In vivo results demonstrated that these cysteine residues are reduced in the periplasm of Hg(2+)-resistant Escherichia coli. Denaturation and redox equilibrium studies revealed that reduced MerP is thermodynamically favored over the oxidized form. The relative stability of reduced MerP appears to be related to the lowered thiol pK(a) (5.5) of the Cys-17 side chain. Despite its much lower pK(a), the Cys-17 thiol is far less accessible than Cys-14, reacting 45 times more slowly with iodoacetamide at pH 7.5. This is reminiscent of proteins such as thioredoxin and DsbA, which contain a similar C-X-X-C motif, except in those cases the more exposed thiol has the lowered pK(a). In terms of MerP function, electrostatic attraction between Hg(2+) and the buried Cys-17 thiolate may be important for triggering the structural change that MerP has been reported to undergo upon Hg(2+) binding. Control of cysteine residue reactivity in heavy metal binding motifs may generally be important in influencing specific metal-binding properties of proteins containing them.     

Role of cysteine residues in the lac permease of Escherichia coli

Oligonucleotide-directed, site-specific mutagenesis has been utilized to replace cysteine residues 117, 333, or 353 and 355 with serine in the lac permease of Escherichia coli. Replacement of Cys-117 or Cys-333 has no significant effect on permease activity, while permease with serine residues in place of Cys-353 and Cys-355 has about 50% of wild-type permease activity. The results provide a clear demonstration that cysteine residues at positions 117, 333, 353, and 355 are not obligatory for lactose/H+ symport. When considered in conjunction with previous findings, the results indicate that, of the eight cysteine residues in the lac permease, only Cys-154 is important for lactose transport. As discussed, the conclusion has important implications for the hypothesis that sulfhydryl-disulfide interconversion plays an important role in the symport mechanism.     

Critical spacing between two essential cysteine residues in the interdomain linker of the Bradyrhizobium japonicum NifA protein

A special sequence motif in the Bradyrhizobium japonicum NifA protein, consisting of two functionally essential cysteines separated by four other amino acids (Cys-aa4-Cys), has been proposed to be part of a potential metal-binding site [(1988) Nucleic Acids Res. 16, 2207-2224]. Using the techniques of oligonucleotide-directed mutagenesis, we report here that several of the four intervening amino acids can be replaced by others without loss of NifA function. The deletion of one amino acid to give a Cys-aa3-Cys motif renders the protein inactive whereas the creation of a Cys-aa5-Cys motif (one amino acid inserted) still leads to a partially active NifA protein.