scholarly journals Disulfide Bond Configuration of Human Cytomegalovirus Glycoprotein B

2002 ◽  
Vol 76 (12) ◽  
pp. 6073-6082 ◽  
Author(s):  
Matthew Lopper ◽  
Teresa Compton
Keyword(s):  
Human Cytomegalovirus ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Cysteine Residue ◽  
Multiple Roles ◽  
Glycoprotein B ◽  
Cysteine Residues ◽  
Amino Terminal ◽  
High Degree ◽  
Human Herpesviruses

ABSTRACT 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.

scholarly journals The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis

2007 ◽  
Vol 81 (10) ◽  
pp. 5212-5224 ◽  
Author(s):  
Michael Mach ◽  
Karolina Osinski ◽  
Barbara Kropff ◽  
Ursula Schloetzer-Schrehardt ◽  
Magdalena Krzyzaniak ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Critical Role ◽  
Point Mutations ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Type I ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Assembly Compartment

ABSTRACT Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.

scholarly journals Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) fromNeisseria meningitidis

2012 ◽  
Vol 68 (7) ◽  
pp. 730-737 ◽  
Author(s):  
Sarah Sainsbury ◽  
Jingshan Ren ◽  
Nigel J. Saunders ◽  
David I. Stuart ◽  
Raymond J. Owens
Keyword(s):  
Crystal Structure ◽  
Transcription Factors ◽  
Neisseria Meningitidis ◽  
Disulfide Bond ◽  
Binding Pocket ◽  
Cysteine Residues ◽  
Regulatory Domain ◽  
High Degree ◽  
Effector Binding ◽  
Lysr Family

The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator fromNeisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.

scholarly journals Intra- and Intermolecular Disulfide Bonds of theGP2b Glycoprotein of Equine Arteritis Virus: Relevance forVirus Assembly andInfectivity

2003 ◽  
Vol 77 (24) ◽  
pp. 12996-13004 ◽  
Author(s):  
Roeland Wieringa ◽  
Antoine A. F. de Vries ◽  
Sabine M. Post ◽  
Peter J. M. Rottier
Keyword(s):  
Viral Entry ◽  
Disulfide Bonds ◽  
Rna Virus ◽  
Cysteine Residue ◽  
Envelope Proteins ◽  
Equine Arteritis Virus ◽  
Cysteine Residues ◽  
Virus Infectivity ◽  
Particle Assembly ◽  
Positive Strand Rna

ABSTRACT Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV virions contain six different envelope proteins. The glycoprotein GP5 (previously named GL) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP2b (previously named GS), GP3, and GP4 are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP5 and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP2b, GP3, and GP4 proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP2b protein in the assembly of the GP2b/GP3/GP4 complex. Open reading frames encoding cysteine-to-serine mutants of the GP2b protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP2b forms an intermolecular cystine bridge with one of the cysteines of the GP4 protein, while the cysteine residues at positions 48 and 137 of GP2b are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP4 protein is responsible for the covalent association of GP3 with the disulfide-linked GP2b/GP4 heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.

scholarly journals Activity of the Yap1 Transcription Factor in Saccharomyces cerevisiae Is Modulated by Methylglyoxal, a Metabolite Derived from Glycolysis

2004 ◽  
Vol 24 (19) ◽  
pp. 8753-8764 ◽  
Author(s):  
Kazuhiro Maeta ◽  
Shingo Izawa ◽  
Shoko Okazaki ◽  
Shusuke Kuge ◽  
Yoshiharu Inoue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Disulfide Bond ◽  
Target Genes ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Glyoxalase I ◽  
Toxic Metabolite ◽  
Basic Leucine Zipper

ABSTRACT Methylglyoxal (MG) is synthesized during glycolysis, although it inhibits cell growth in all types of organisms. Hence, it has long been asked why such a toxic metabolite is synthesized in vivo. Glyoxalase I is a major enzyme detoxifying MG. Here we show that the Yap1 transcription factor, which is critical for the oxidative-stress response in Saccharomyces cerevisiae, is constitutively concentrated in the nucleus and activates the expression of its target genes in a glyoxalase I-deficient mutant. Yap1 contains six cysteine residues in two cysteine-rich domains (CRDs), i.e., three cysteine residues clustering near the N terminus (n-CRD) and the remaining three cysteine residues near the C terminus (c-CRD). We reveal that any of the three cysteine residues in the c-CRD is sufficient for MG to allow Yap1 to translocate into the nucleus and to activate the expression of its target gene. A Yap1 mutant possessing only one cysteine residue in the c-CRD but no cysteine in the n-CRD and deletion of the basic leucine zipper domain can concentrate in the nucleus with MG treatment. However, substitution of all the cysteine residues in Yap1 abolishes the ability of this transcription factor to concentrate in the nucleus following MG treatment. The redox status of Yap1 is substantially unchanged, and protein(s) interaction with Yap1 through disulfide bond is hardly detected in cells treated with MG. Collectively, neither intermolecular nor intramolecular disulfide bond formation seems to be involved in Yap1 activation by MG. Moreover, we show that nucleocytoplasmic localization of Yap1 closely correlates with growth phase and intracellular MG level. We propose a novel regulatory pathway underlying Yap1 activation by a natural metabolite in the cell.

Identification of Cysteine Residues Essential for von Willebrand Factor (VWF) Multimerization.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1799-1799
Author(s):  
Angie R. Purvis ◽  
Julia Gross ◽  
Luke T. Dang ◽  
Ren-Huai Huang ◽  
Milan Kapadia ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Expression System ◽  
Quadrupole Ion Trap ◽  
Cysteine Residues ◽  
Ion Cyclotron Resonance ◽  
Unbound Fraction ◽  
Bhk Cells ◽  
Interchain Disulfide Bond ◽  
Doubly Charged

Abstract VWF assembles into multimers in the Golgi by forming disulfide bonds between adjacent D’D3 domains using an oxidoreductase mechanism that requires the VWF propeptide (D1D2). During multimerization, free thiols are predicted to be important in the oxidoreductase mechanism either as the cysteine residues that form interchain disulfide bonds in the VWF multimer or as the cysteine residues that rearrange the disulfide-bonded intermediate between the propeptide and D’D3. To characterize free thiols involved in VWF multimerization, a model construct containing domains D1D2D’D3 was expressed in baby hamster kidney (BHK) cells and the secreted products were alkylated with thiol-reactive biotin-maleimide reagents and purified on avidin agarose. Both the D’D3 dimer and the D1D2 remained in the avidin agarose unbound fraction, indicating the absence of reduced Cys residues. The D’D3 monomer was bound to the avidin agarose beads, indicating the presence of reduced and alkylated cysteines. The reduced cysteines in the D’D3 monomer were alkylated with N-ethylmaleimide (NEM) for identification by mass spectrometry. NEM-labeled D’D3 monomer was reduced and alkylated with 4-vinylpyridine (4-VP). NEM increases the mass of Cys to 228.05686 Da, while 4-VP shifts the mass of Cys to 208.06703 Da. Peptides were generated with various proteases and analyzed by a linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTMS). A theoretical peptide list was generated including either NEM or 4-VP modification of all Cys residues. The mass spectra were analyzed for m/z signals including up to the triply charged species of the theoretical digest. The MS/MS spectra were searched against a database with MASCOT. Using this method 98% of the 52 Cys residues within the D’D3 region were identified as either 4-VP- or NEM-modified. Cys1142 was identified in a tryptic peptide (ENGYECEWR+NEM, VWF residues 1137–1145) as a doubly charged ion at m/z 655.76031, confirming the previous identification of the Cys1142-Cys1142 interchain disulfide bond linking VWF multimers. A second NEM-modified peptide was identified from an Asp-N protease digest (DCACFC+2 4-VP+1 NEM, residues 1096–1101 of VWF). This species was observed as a doubly charged ion at m/z 498.67584. Analysis of the MS/MS revealed that Cys1099 was NEM-modified, suggesting that it may form an interchain disulfide bond between VWF multimers or may be important in the oxidoreductase mechanism of VWF multimerization. To determine if C1099 and/or C1142 are required to form D’D3 dimers, constructs D1D2D’D3 (C1099A), (C1142A), and (C1099A/C1142A) were expressed in BHK cells and the assembly of D’D3 dimers was assayed by Western blotting. All three mutant VWF proteins were secreted efficiently from BHK cells. However, the levels of D’D3 dimer were reduced markedly in the single mutants when compared with the dimer produced by the wild type D1D2D’D3 expression system. Further, mutation of both C1142 and C1099 (C1099A/C1142A) completely inhibited D’D3 dimerization, evidence that these specific cysteine residues in the D’D3 monomer are involved in the interchain disulfide bonds between VWF multimers and are essential for the oxidoreductase mechanism of VWF multimerization.

Two dileucine motifs mediate late endosomal/lysosomal targeting of transmembrane protein 192 (TMEM192) and a C-terminal cysteine residue is responsible for disulfide bond formation in TMEM192 homodimers

2011 ◽  
Vol 434 (2) ◽  
pp. 219-231 ◽  
Author(s):  
Jörg Behnke ◽  
Eeva-Liisa Eskelinen ◽  
Paul Saftig ◽  
Bernd Schröder
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Transmembrane Protein ◽  
Bond Formation ◽  
Immunogold Labelling ◽  
Disulfide Bridge ◽  
Cysteine Residue ◽  
Disulfide Bond Formation ◽  
Transmembrane Segments ◽  
Late Endosomes

TMEM192 (transmembrane protein 192) is a novel constituent of late endosomal/lysosomal membranes with four potential transmembrane segments and an unknown function that was initially discovered by organellar proteomics. Subsequently, localization in late endosomes/lysosomes has been confirmed for overexpressed and endogenous TMEM192, and homodimers of TMEM192 linked by disulfide bonds have been reported. In the present study the molecular determinants of TMEM192 mediating its transport to late endosomes/lysosomes were analysed by using CD4 chimaeric constructs and mutagenesis of potential targeting motifs in TMEM192. Two directly adjacent N-terminally located dileucine motifs of the DXXLL-type were found to be critical for transport of TMEM192 to late endosomes/lysosomes. Whereas disruption of both dileucine motifs resulted in mistargeting of TMEM192 to the plasma membrane, each of the two motifs was sufficient to ensure correct targeting of TMEM192. In order to study disulfide bond formation, mutagenesis of cysteine residues was performed. Mutation of Cys266 abolished disulfide bridge formation between TMEM192 molecules, indicating that TMEM192 dimers are linked by a disulfide bridge between their C-terminal tails. According to the predicted topology, Cys266 would be localized in the reductive milieu of the cytosol where disulfide bridges are generally uncommon. Using immunogold labelling and proteinase protection assays, the localization of the N- and C-termini of TMEM192 on the cytosolic side of the late endosomal/lysosomal membrane was experimentally confirmed. These findings may imply close proximity of the C-termini in TMEM192 dimers and a possible involvement of this part of the protein in dimer assembly.

scholarly journals Stepwise Oxidations Play Key Roles in the Structural and Functional Regulations of DJ-1

2021 ◽  
Author(s):  
In-Kang Song ◽  
Mi-Sun Kim ◽  
James E. Ferrell ◽  
Dong-Hae Shin ◽  
Kong-Joo Lee
Keyword(s):  
Mass Spectrometry ◽  
Disulfide Bond ◽  
Antioxidant Activities ◽  
Neuronal Cell ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Disulfide Crosslinking ◽  
Functional Changes ◽  
Exchange Mass Spectrometry ◽  
Dopaminergic Neuronal Cell

AbstractDJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. We found that Cys46 is also reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe that selectively reacts with redox sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. We also found that Cys46-Cys53 disulfide crosslinking affects the oxidative state of the third Cys106, which shows the crosstalk among three cysteine residues of DJ-1. Furthermore, we demonstrated that DJ-1 C46A mutant, not forming Cys46-Cys53 intra-disulfide bond, lost structural stability of DJ-1 employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike wild type DJ-1. These findings suggest that DJ-1 regulates its structure and activities by concerted oxidative modifications of three cysteine residues. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.

Sequence variation of the amino-terminal antigenic domains of glycoprotein B of human cytomegalovirus strains isolated from Chinese patients

1994 ◽  
Vol 137 (1-2) ◽  
pp. 133-138 ◽  
Author(s):  
S. Y. W. Shiu ◽  
K. M. Chan ◽  
S. K. F. Lo ◽  
K. W. Y. Ip ◽  
K. Y. Yuen ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Sequence Variation ◽  
Chinese Patients ◽  
Glycoprotein B ◽  
Amino Terminal ◽  
Antigenic Domains

scholarly journals The amino acid sequence around four cysteine residues in trout actin

1971 ◽  
Vol 123 (4) ◽  
pp. 591-600 ◽  
Author(s):  
John Bridgen
Keyword(s):  
Acetic Acid ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cysteine Residue ◽  
Amino Acid Sequences ◽  
Cysteine Residues ◽  
Muscle Actin ◽  
Guanidinium Chloride ◽  
High Degree ◽  
Trout Muscle

Four unique carboxymethylcysteine-containing peptides were isolated from tryptic and chymotryptic digests of trout muscle actin carboxymethylated with iodo[2-14C]acetic acid in 6m-guanidinium chloride. The amino acid sequences of these peptides were determined and showed a high degree of homology with the corresponding sequences from rabbit actin. One of the radioactive peptides was the C-terminal peptide and another sequence probably contained the cysteine residue from the N-terminal region of the protein.

scholarly journals Human Cytomegalovirus Elicits a Coordinated Cellular Antiviral Response via Envelope Glycoprotein B

2004 ◽  
Vol 78 (3) ◽  
pp. 1202-1211 ◽  
Author(s):  
Karl W. Boehme ◽  
Jasbir Singh ◽  
Stuart T. Perry ◽  
Teresa Compton
Keyword(s):  
Human Cytomegalovirus ◽  
Envelope Glycoprotein ◽  
Cell Types ◽  
Antiviral Effect ◽  
Antiviral Response ◽  
Cell Surface Receptor ◽  
Glycoprotein B ◽  
Antiviral State ◽  
Amino Terminal ◽  
Interferon Pathway

ABSTRACT Previous studies have shown that human cytomegalovirus (CMV) is a potent elicitor of interferon-stimulated gene (ISG) expression. Induction of the interferon pathway does not require replication-competent virus, and envelope glycoprotein B (gB) from CMV is a viral structural component that can directly induce transcription of ISGs. Here we extend these earlier findings by defining the consequences of inducing the interferon pathway. We found that cells respond to CMV or soluble gB by establishing a functional antiviral state within cell types critical in CMV biology, such as fibroblasts and endothelial cells. We have also discovered new insights into the mechanism by which the pathway is initiated. Interferon regulatory factor 3 (IRF3), a key transcriptional regulator of cellular interferon responses, is activated by CMV virions and soluble gB. Thus, IRF3 becomes activated via “outside-in” signal transduction events. This is a novel mechanism of activation of this key transcription factor by viruses. In comparison to soluble gB (gB1-750), which comprises the entire ectodomain of gB, a truncation mutant encompassing only the amino-terminal region of gB (gB1-460) was markedly less effective at inducing antiviral responses. This indicates that the region of gB from residues 461 to 750 is important for initiation of the antiviral response. In addition, CMV and gB establish an antiviral state in alpha/beta interferon null cells, illustrating that primary induction of ISGs by CMV and gB is sufficient to establish the antiviral response and that interferon secretion is not necessary for the antiviral effect. Taken together, our findings reveal that CMV initiates a coordinated antiviral response through contact between gB and an as-yet-unidentified cell surface receptor(s).

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