Molecular mechanism of oxidative stress perception by the Orp1 protein

In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H(2)O(2) Orp1(Cys36) forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys(36) of Orp1(Cys36) upon exposure to H(2)O(2). Under similar conditions, neither Cys(82) of Orp1(Cys82) nor Cys(598) of Yap1 forms Cys-SOH. A homology-based molecular model of Orp1 suggests that the structure of the active site of Orp1 is similar to that found in mammalian selenocysteine glutathione peroxidases. Proposed active site residues Gln(70) and Trp(125) form a catalytic triad with Cys(36) in the Orp1 molecular model. The remainder of the active site pocket is formed by Phe(38), Asn(126), and Phe(127), which are evolutionarily conserved residues. We made Q70A and W125A mutants and tested the ability of these mutants to form Cys-SOH in response to H(2)O(2). Both mutants were unable to form Cys-SOH and did not form a H(2)O(2)-inducible disulfide-bonded complex with Yap1-cCRD. The pK(a) of Cys(36) was determined to be 5.1, which is 3.2 pH units lower than that of a free cysteine (8.3). In contrast, Orp1 Cys(82) (the resolving cysteine) has a pK(a) value of 8.3. The pK(a) of Cys(36) in the Q70A and W125A mutants is also 8.3, demonstrating the importance of these residues in modulating the nucleophilic character of Cys(36). Finally, we show that S. cerevisiae strains with ORP1 Q70A and W125A mutations are less tolerant to H(2)O(2) than those containing wild-type ORP1. The results of our study suggest that attempts to identify novel redox-regulated proteins and signal transduction pathways should focus on characterization of low pK(a) cysteines.     

Isd11p protein activates the mitochondrial cysteine desulfurase Nfs1p protein

Cysteine desulfurases perform pyridoxal phosphate (PLP)-dependent desulfuration of cysteine. The key steps of the enzymatic cycle include substrate binding to PLP, formation of a covalent persulfide intermediate at the active site cysteine, and transfer of sulfur to recipients for use in various metabolic pathways. In Saccharomyces cerevisiae, the cysteine desulfurase Nfs1p and an accessory protein, Isd11p, are found primarily in mitochondria, and both are essential for cell viability. Although cysteine desulfurases are conserved from bacteria to humans, Isd11p is found only in eukaryotes and not in prokaryotes. Here we show that Isd11p activates Nfs1p. The enzyme without Isd11p was inactive and did not form the [(35)S]persulfide intermediate from the substrate [(35)S]cysteine. Addition of Isd11p to inactive Nfs1p induced formation of the persulfide. Remarkably, in a two-step assay, [(35)S]cysteine could be bound to the inactive Nfs1p in a PLP-dependent manner, and the enzyme could be subsequently induced to form the persulfide by addition of Isd11p. A mutant form of Isd11p with the (15)LYK(17) motif changed to (15)AAA(17) was able to bind but failed to activate Nfs1p, thus separating these two functions of Isd11p. Finally, compared with Nfs1p with or without the bound Isd11p mutant, the Nfs1p·Isd11p complex was more resistant to inactivation by an alkylating agent. On the basis of these novel findings, we propose that interaction of Isd11p with Nfs1p activates the enzyme by inducing a conformational change, thereby promoting formation of the persulfide intermediate at the active site cysteine. Such a conformational change may protect the active site cysteine from alkylating agents.     

Mutagenesis and derivatization of human vesicle monoamine transporter 2 (VMAT2) cysteines identifies transporter domains involved in tetrabenazine binding and substrate transport

The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. Photoaffinity labeling, chimera analysis, and mutagenesis have identified functionally important amino acids and provided some information regarding structure and ligand binding sites. To extend these studies, we engineered functional human VMAT2 constructs with reduced numbers of cysteines. Subsets of cysteines were discovered, which restore function to an inactive cysteine-less human VMAT2. Replacement of three transmembrane (TM) cysteines together (net removal/replacement of three atoms) significantly enhanced monoamine transport. Cysteine modification studies involving single and combination cysteine mutants with methanethiosulfonate ethylamine revealed that [(3)H]dihydrotetrabenazine binding is > 90% inhibited by modification of two sets of cysteines. The primary target (responsible for approximately 80% of inhibition) is Cys(439) in TM 11. The secondary target (responsible for approximately 20% of inhibition) is one or more of the four non-TM cysteines. [(3)H]Dihydrotetrabenazine protects against modification of Cys(439) by a 10,000-fold molar excess of methanethiosulfonate ethylamine, demonstrating that Cys(439) is either at the tetrabenazine binding site, or conformationally linked to tetrabenazine binding. Supporting a direct effect, the position of tetrabenazine-protectable Cys 439 is consistent with previous mutagenesis, chimera, and photoaffinity labeling data, demonstrating involvement of TM 10-12 in a tetrabenazine binding domain.     

p36, the major cytoplasmic substrate of src tyrosine protein kinase, binds to its p11 regulatory subunit via a short amino-terminal amphiphatic helix.

Protein I is a hetero-tetramer which contains two copies each of p11 and p36. p36 (calpactin I, lipocortin II) is a major substrate of retrovirally encoded tyrosine protein kinases, while p11 modulates several Ca2+-induced properties also displayed by p36 alone. Here we have characterized the p11 binding site on p36 by fluorescence spectroscopy using porcine p36 labelled at cysteine 8 with the fluorophore Prodan (6-proprionyl-2-dimethylamino-naphthalene). We have used peptides of differing length from the amino-terminal domain of p36 to restrict the major binding site to the first 12 residues. Noticeable binding is still observed with a peptide containing only the first nine residues. Interestingly the N-terminal acetyl group of p36 forms a functional part of the p11 binding site. CD studies indicate that the binding region can form an alpha-helix, which seems to have amphiphatic properties when projected on a helical wheel. This structural element is also known for a calmodulin binding protein. Thus the question is raised whether other p11/calmodulin-related proteins interact with their target proteins via a similar mechanism. We also discuss how p11 could modulate p36 associated properties.     

The polyamine binding site in inward rectifier K+ channels

Strongly inwardly rectifying potassium channels exhibit potent and steeply voltage-dependent block by intracellular polyamines. To locate the polyamine binding site, we have examined the effects of polyamine blockade on the rate of MTSEA modification of cysteine residues strategically substituted in the pore of a strongly rectifying Kir channel (Kir6.2[N160D]). Spermine only protected cysteines substituted at a deep location in the pore, between the "rectification controller" residue (N160D in Kir6.2, D172 in Kir2.1) and the selectivity filter, against MTSEA modification. In contrast, blockade with a longer synthetic polyamine (CGC-11179) also protected cysteines substituted at sites closer to the cytoplasmic entrance of the channel. Modification of a cysteine at the entrance to the inner cavity (169C) was unaffected by either spermine or CGC-11179, and spermine was clearly "locked" into the inner cavity (i.e., exhibited a dramatically slower exit rate) following modification of this residue. These data provide physical constraints on the spermine binding site, demonstrating that spermine stably binds at a deep site beyond the "rectification controller" residue, near the extracellular entrance to the channel.     

Mutating the four extracellular cysteines in the chemokine receptor CCR6 reveals their differing roles in receptor trafficking,ligand binding,and signaling

CCR6 is the receptor for the chemokine MIP-3 alpha/CCL20. Almost all chemokine receptors contain cysteine residues in the N-terminal domain and in the first, second, and third extracellular loops. In this report, we have studied the importance of all cysteine residues in the CCR6 sequence using site-directed mutagenesis and biochemical techniques. Like all G protein-coupled receptors, mutating disulfide bond-forming cysteines in the first (Cys118) and second (Cys197) extracellular loops in CCR6 led to complete elimination of receptor activity, which for CCR6 was also associated with the accumulation of the receptor intracellularly. Although two additional cysteines in the N-terminal region and the third extracellular loop, which are present in almost all chemokine receptors, are presumed to form a disulfide bond, this has not been demonstrated experimentally for any of these receptors. We found that mutating the cysteines in the N-terminal domain (Cys36) and the third extracellular loop (Cys288) neither significantly affected receptor surface expression nor completely abolished receptor function. Importantly, contrary to several previous reports, we demonstrated directly that instead of forming a disulfide bond, the N-terminal cysteine (Cys36) and the third extracellular loop cysteine (Cys288) contain free SH groups. The cysteine residues (Cys36 and Cys288), rather than forming a disulfide bond, may be important per se. We propose that CCR6 forms only a disulfide bond between the first (Cys118) and second (Cys197) extracellular loops, which confines a helical bundle together with the N-terminus adjacent to the third extracellular loop, creating the structural organization critical for ligand binding and therefore for receptor signaling.     

Functionally distinct serine phosphorylation sites of p36, the cellular substrate of retroviral protein kinase; differential inhibition of reassociation with p11.

P36 was originally defined as the major cytoplasmic target of retrovirally coded tyrosine-kinases. While recently much has been learned about its biochemistry, the functional importance of its tyrosine and serine phosphorylation has not been approached. As p36 is now understood as a multi-ligand protein its in vitro phosphorylation by three different serine/threonine kinases was followed. Monomeric p36 is a much better substrate than the complex containing two copies each of p36 and p11 (protein I). All p36 phosphorylation sites occur within the amino-terminal 29 residues specifically released by mild proteolysis. As this region harbors an important interaction site for p11 the reduced phosphorylation of p36 in the protein I complex results most likely from a lowered accessibility. Phosphorylation of p36 is serine specific. Reconstitution experiments define at least two functionally distinct sites. One product of protein kinase C reconstitutes with p11 to protein I, while this complex formation normal for p36 is observed neither for the second phosphorylation product nor for the derivatives resulting from phosphorylation by calmodulin or cAMP dependent kinases. The results lend direct support to the hypothesis that phosphorylation of p36 can modulate one of its molecular functions. Obvious implications for other Ca2+-dependent lipid binding proteins are discussed.     

Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins

Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin-proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p.     

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.     

Differential roles played by the native cysteine residues of the yeast glutathione transporter, Hgt1p

Hgt1p, a high-affinity glutathione transporter from the yeast Saccharomyces cerevisiae , belongs to the structurally uncharacterized oligopeptide transporter (OPT) family. To initiate structural studies on Hgt1p, a cysteine-free (cys-free) Hgt1p was generated. This cys-free Hgt1p was nonfunctional and pointed to a critical role being played by the native cysteine residues of Hgt1p. To investigate their role, genetic and biochemical approaches were undertaken. Functional suppressors of the cys-free Hgt1p were isolated, and yielded double revertants bearing C622 and C632. Subsequent biochemical characterization of the individual C622S/A or C632S/A mutations revealed that both these cysteine residues were, in fact, individually indispensable for Hgt1p function and were required for trafficking to the plasma membrane. However, despite their essentiality, the presence of only these two native cysteines in Hgt1p generated a very weak glutathione transporter with minimal functional activity. Hence, the remaining 10 cysteines were also contributing towards Hgt1p activity, although they were not found to be singly responsible or crucial for Hgt1p functional activity. These residues, however, contributed cumulatively towards the stability and the functionality of Hgt1p, without affecting the trafficking to the cell surface. The study reveals differential roles for the cysteines of Hgt1p and provides first insights into the structural features of an OPT family member.     

New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively. PEX26 responsible for peroxisome biogenesis disorders of complementation group 8 codes for C-tail-anchored type-II membrane peroxin Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by ATPase cycle. Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome targeting signal type-1 and shuttles between the cytosol and peroxisomes. AAA peroxins are involved in the export from peroxisomes of Pex5p. Pex5p is ubiquitinated at the conserved cysteine11 in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p-C11A, abrogates peroxisomal import of proteins harboring peroxisome targeting signals 1 and 2 in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, hence suggesting an essential role of the cysteine residue in the export of Pex5p.     

Alanine scanning of all cysteines and construction of a functional cysteine-less Cdr1p, a multidrug ABC transporter of Candida albicans

Herein, we discuss the role of the native cysteines present in a major multidrug ABC transporter of Candida albicans, Cdr1p, and describe the construction of this transporter's functional cysteine-less (cysless) protein version for cross-linking studies. In the experiments in which all 23 cysteines were replaced individually, we observed that most of the cysteine replacements were tolerated by the protein, but the replacement of C1056, C1091, C1106, C1294 or C1336 resulted in an enhanced drug susceptibility together with an abrogated drug efflux. Notably, the ATPase activity was uncoupled, which largely remained unaffected in these variants. The substitution of the critical cysteines with serines restored the normal expression and functionality of Cdr1p because serine can effectively mimic the hydrogen bonding properties of cysteine. Finally, we constructed a functional cysless His-tagged Cdr1p in which all the cysteines of the native protein were replaced with alanines and the critical cysteines were replaced with serines. Notably, cysless GFP-tagged variant of Cdr1p was non-functional. The cysless His-tagged variant of Cdr1p is the first example of a cysless ABC transporter in yeast, and it will lead to a greater understanding of the architecture of this important protein and provide insight into the nature of drug binding and interdomain communication.     

Protein-protein interaction studied by site-directed mutagenesis. Characterization of the annexin II-binding site on p11, a member of the S100 protein family.

p11, a member of the S100 protein family, forms a stable heterotetrameric complex with annexin II. The p11-binding site of annexin II resides in the N-terminal 14 residues, which form an amphiphatic alpha-helix with the hydrophobic face representing the contact site for p11 (Johnsson, N., Marriott, G., and Weber, K. (1988) EMBO J. 7, 2435-2442). We show that a corresponding peptide can be used to purify recombinant p11 by affinity chromatography. To map the annexin II-binding site on p11, we have produced progressively truncated p11 derivatives by site-directed mutagenesis. Our analysis reveals that a highly hydrophobic region between residues 85 and 91 is indispensable for annexin II-binding. It is located in the C-terminal extension, following the second distorted EF-hand. Using a series of single amino acid replacements, we have identified individual hydrophobic residues, which seem to represent contact points for annexin II. Most notably, substitution of tyrosine 85 or phenylalanine 86 by alanine drastically reduces the affinity of p11 for annexin II, whereas replacement of these residues by tryptophan has no or only a marginal effect. Thus, hydrophobic side chains on both annexin II and p11 are involved in complex formation.     

Transport function of the renal type IIa Na+/P(i) cotransporter is codetermined by residues in two opposing linker regions

Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/P(i) cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region. The effect of cysteine modification by different MTS reagents on two electrogenic transport modes (leak and cotransport) was investigated. MTSEA (2-aminoethyl MTS hydrobromide) and MTSES (MTS ethylsulfonate) led to full inhibition of cotransport and increased the leak, whereas incubation in MTSET (2-[trimethylammonium]ethyl MTS bromide) inhibited only cotransport. The behavior of other double mutants with a cysteine retained at one site and hydrophobic or hydrophilic residues substituted at the other site, indicated that most likely only Cys-460 was modifiable, but the residue at Ala-203 was critical for conferring the leak and cotransport mode behavior. Substrate interaction with the double mutant was unaffected by MTS exposure as the apparent P(i) and Na+ affinities for P(i)-induced currents and respective activation functions were unchanged after cysteine modification. This suggested that the modified site did not interfere with substrate recognition/binding, but prevents translocation of the fully loaded carrier. The time-dependency of cotransport loss and leak growth during modification of the double cysteine mutant was reciprocal, which suggested that the modified site is a kinetic codeterminant of both transport modes. The behavior is consistent with a kinetic model for NaPi-IIa that predicts mutual exclusiveness of both transport modes. Together, these findings suggest that parts of the opposing linker regions are associated with the NaPi-IIa transport pathway.     

Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.     

S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection,in vitro reconstitution and role in viral infectivity

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.