MC4R oligomerizes independently of extracellular cysteine residues

The melanocortin 4 receptor (MC4R) plays an essential role in weight regulation. Recently we could show that the MC4R is able to form receptor dimers. In the present study we investigated the role of extracellular cysteine residues and the structure of the third extracellular loop for receptor dimerization. None of the four extracellular cysteine residues nor the structure of the third extracellular loop play a role for MC4R-MC4R interaction as all investigated mutants display the same dimerization pattern as the wild-type receptor. Therefore for MC4R dimerization structures of the transmembrane-spanning helices are more likely to be involved.     

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.     

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.     

Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition

The epithelial Na(+) channel (ENaC) is typically formed by three homologous subunits (alpha, beta, and gamma) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na(+) self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na(+). All 16 Cys residues within alpha and gamma ECLs and selected beta ECL Cys residues were individually mutated to alanine or serine residues. The Na(+) self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight alpha (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one beta (Cys-7), and nine gamma (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na(+) self-inhibition. Na(+) self-inhibition was eliminated by simultaneous mutations of either the last three alpha ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both alpha and gamma ECLs. By analyzing the Na(+) self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the alphaECL (alphaCys-1/alphaCys-6, alphaCys-4/alphaCys-5, alphaCys-7/alphaCys-16, alphaCys-10/alphaCys-13, and alphaCys-11/alphaCys-12) and one pair within the gammaECL (gammaCys-7/gammaCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the alpha and gamma ENaC subunits are required to establish the tertiary structure that ensures a proper Na(+) self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.     

Reexamination of the cysteine residues in glucocerebrosidase

Glucocerebrosidase, the deficient enzyme in Gaucher disease, catalyzes the cleavage of the beta-glycosidic linkage of glucosylceramide. A previous study on the enzyme identified three disulfide bridges and a single sulfhydryl [Lee, Y., Kinoshita, H., Radke, G., Weiler, S., Barranger, J.A. and Tomich, J.M. (1995) Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase. J. Protein Chem. 14(3), 127-137] but recent publication of the X-ray structure identifies only two disulfide bridges with three free sulfhydryls [Dvir, H., Harel, M., McCarthy, A.A., Toker, L., Silman, I., Futerman, A.H. and Sussman, J.L. (2003) X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO. 4(7), 704-709]. Using chemical modifications, acid cleavage and enzymatic digestion methods, we report that three free sulfhydryls exist and that the remaining four cysteines form two disulfide bonds located within the first 25 amino-terminal residues, supporting the X-ray structure.     

Catalytic cysteine residues of ER-60 protease

ER-60 protease contains two CGHC motifs that appear to include an active site cysteine residue(s). Its proteolytic activity was lost with a double mutation of the C-terminal cysteines of the two motifs to alanine, but not with a single mutation of the C-terminal cysteine of either of the motifs to alanine. This suggests that these C-terminal cysteines independently constitute the catalytic active site. A mutation of both histidine residues in the two CGHC motifs to serine did not abolish the proteolytic activity, suggesting these histidine residues in the CGHC motifs do not constitute the catalytic dyad of ER-60 protease.     

Role of extracellular cysteine residues in the adenosine A<Subscript>2A</Subscript> receptor

The G protein-coupled A_2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71^2.69-Cys159^45.43; II, Cys74^3.22-Cys146^45.30, and III, Cys77^3.25-Cys166^45.50). However, the A_2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A_2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A_2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A_2A and A_2BARs.     

Highly reactive cysteine residues in rodent hemoglobins

Two hemoglobins with cysteine residues highly reactive toward electrophiles have been identified and characterized. Cys-125beta of guinea pig hemoglobin has a low pK(a) and forms conjugates with electrophiles more quickly than glutathione and several orders of magnitude more quickly than other protein thiols. This cysteine is capable of intercepting benzoquinone, a known carcinogenic metabolite, before other protein nucleophiles can be modified. Cys-13beta of mouse hemoglobin was observed to conjugate with electrophiles as quickly as glutathione. The structural basis of reactivity is different in the two hemoglobins and is analyzed in terms of hydrogen-bonding, solvent accessibility, and helix-dipole contributions. Complementing a previously characterized highly reactive cysteine in rat hemoglobin, identification of these cysteines suggests that the reactivity of these hemoglobins could represent a common function as a detoxification sink against carcinogens.     

Novel oxidative modifications in redox-active cysteine residues

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.     

Mutational analysis of roles for extracellular cysteine residues in the assembly and function of human alpha 7-nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) containing alpha7 subunits self-assemble into simple, homopentameric complexes. However, successful heterologous expression of functional alpha7-nAChR has only been achieved in a few host cell types, such as the SH-EP1 human epithelial cell line. All ionotropic glycine receptor, GABA(A) receptor, 5-HT(3) receptor, and nAChR subunits contain a pair of highly conserved cysteine residues (C150 and C164 for alpha7 subunits) in their N-terminal extracellular domain. These residues are thought to be involved in the formation of a conserved cystine loop that is critical to the proper folding and assembly of subunits. However, nAChR alpha7 (and alpha8) subunits also contain a third cysteine residue, C138, N-terminal to the conserved cysteine pair. Using SH-EP1 cells as a host for heterologous expression, we evaluated the roles of C138, C150, and C164 in subunit folding, assembly, and cell surface expression and function of alpha7-nAChR. Results indicate that mutation of C138, but not of C150 or C164, yields an nAChR that can assemble to form (125)I-labeled alpha-bungarotoxin binding sites expressed on the cell surface. Further, whole-cell patch clamp recordings demonstrate that mutation of C138 to alanine does not alter the function of the fully assembled alpha7-nAChR. These results indicate that C150 and C164 are required for surface expression, but that C138 is neither necessary for nor inhibitory toward the surface expression and function of human alpha7-nAChR. These results suggest that disulfide bond formation between C138 and either C150 or C164, if it occurs, has no significant effect on alpha7-nAChR assembly or function.     

Analysis and functional prediction of reactive cysteine residues

Cys is much different from other common amino acids in proteins. Being one of the least abundant residues, Cys is often observed in functional sites in proteins. This residue is reactive, polarizable, and redox-active; has high affinity for metals; and is particularly responsive to the local environment. A better understanding of the basic properties of Cys is essential for interpretation of high-throughput data sets and for prediction and classification of functional Cys residues. We provide an overview of approaches used to study Cys residues, from methods for investigation of their basic properties, such as exposure and pK(a), to algorithms for functional prediction of different types of Cys in proteins.     

The cysteine residues of recombinant human gastric lipase

Recombinant human gastric lipase (rHGL) and three of its cysteine mutants (cysteine 227, 236, and 244 substitued for threonine or serine) were expressed in the baculovirus/insect cell system and purified to homogeneity by performing a two-step procedure. Substituting Ser for Cys 227 and Cys 236 resulted in mutant lipases with a significantly lower level of activity (30% and 22%, respectively) on a short chain triglyceride (tribuyrin) substrate, while the mutation at position 244 only slightly reduced the activity. Using 4, 4'-dithiopyridine (4-PDS) as a sulfhydryl reagent on the above mutants, it was possible to clearly identify the single sulfhydryl residue at position 244 and consequently, the disulfide bridge at position 227-236. No potential disulfide bridges were formed during the protein folding between cysteines 227-244 or between cysteines 236-244, as thought to occur in the case of rabbit gastric lipase (RGL). The present results are consistent with the recently determined 3D-structure of rHGL.     

The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residues in ficin

Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and alpha-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined.     

Structural roles of cysteine 50 and cysteine 230 residues in Arabidopsis thaliana S-adenosylmethionine decarboxylase

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA (GenBank U63633) was cloned. Site-specific mutagenesis was performed to introduce mutations at the conserved cysteine Cys(50), Cys(83), and Cys(230), and lys(81) residues. In accordance with the human AdoMetDC, the C50A and C230A mutagenesis had minimal effect on catalytic activity, which was further supported by DTNB-mediated inactivation and reactivation. However, unlike the human AdoMetDC, the Cys(50) and Cys(230) mutants were much more thermally unstable than the wild type and other mutant AdoMetDC, suggesting the structural significance of cysteines. Furthermore, according to a circular dichroism spectrum analysis, the Cys(50) and Cys(230) mutants show a higher a-helix content and lower coiled-coil content when compared to that of wild type and the other mutant AdoMetDC. Also, the three-dimensional structure of Arabidopsis thaliana AdoMetDC could further support all of the data presented here. Summarily, we suggest that the Cys(50) and Cys(230) residues are structurally important.     

Binding of antimitotic drugs around cysteine residues of tubulin

Two independent approaches provide evidence of cysteine residues in the vicinity of the binding sites of colchicine and vinblastine to tubulin: (1) The reactive bromoacetamide group of the affinity label bromocolchicine covalently binds to cysteine residues of tubulin; (2) vinblastine and colchicine slow down the reaction of DTNB with SH groups of tubulin.     

Adjacent cysteine residues as a redox switch.

Oxidation of adjacent cysteine residues into a cystine forms a strained eight-membered ring. This motif was tested as the basis for an enzyme with an artificial redox switch. Adjacent cysteine residues were introduced into two different structural contexts in ribonuclease A (RNase A) by site-directed mutagenesis to produce the A5C/A6C and S15C/S16C variants. Ala5 and Ala6 are located in an alpha-helix, whereas Ser15 and Ser16 are located in a surface loop. Only A5C/A6C RNase A had the desired property. The catalytic activity of this variant decreases by 70% upon oxidation. The new disulfide bond also decreases the conformational stability of the A5C/A6C variant. Reduction with dithiothreitol restores full enzymatic activity. Thus, the insertion of adjacent cysteine residues in a proper context can be used to modulate enzymatic activity.     

Intracellular cysteine residues in the tail of MHC class I proteins are crucial for extracellular recognition by leukocyte Ig-like receptor 1

The activity of NK cells is regulated by activating receptors that recognize mainly stress-induced ligands and by inhibitory receptors that recognize mostly MHC class I proteins on target cells. Comparing the cytoplasmic tail sequences of various MHC class I proteins revealed the presence of unique cysteine residues in some of the MHC class I molecules which are absent in others. To study the role of these unique cysteines, we performed site specific mutagenesis, generating MHC class I molecules lacking these cysteines, and demonstrated that their expression on the cell surface was impaired. Surprisingly, we demonstrated that these cysteines are crucial for the surface binding of the leukocyte Ig-like receptor 1 inhibitory receptor to the MHC class I proteins, but not for the binding of the KIR2DL1 inhibitory receptor. In addition, we demonstrated that the cysteine residues in the cytoplasmic tail of MHC class I proteins are crucial for their egress from the endoplasmic reticulum and for their palmitoylation, thus probably affecting their expression on the cell surface. Finally, we show that the cysteine residues are important for proper extracellular conformation. Thus, although the interaction between leukocyte Ig-like receptor 1 and MHC class I proteins is formed between two extracellular surfaces, the intracellular components of MHC class I proteins play a crucial role in this recognition.     

Specific enzymic cleavage of polypeptides at cysteine residues.

A method has been developed for specific enzymic cleavage of polypeptides at the N-terminal side of modified cysteine residues. Lysine residues are blocked by trifluoroacetylation and cysteine residues subsequently converted to the 2-aminoethyl derivatives. Digestion of the modified polypeptide with the lysine-specific protease from Armillaria mellea (patented by Walton et al., 1972) occurs only at 2-aminoethylcysteine residues. With the beta chain of human haemoglobin, which contains 2 cysteine and 11 lysine residues, cleavage was observed at both modified cysteines but at none of the lysines. In the case of a polypeptide from bee venom which contains 4 half-cystine and 5 lysine residues, cleavage occurred at only 2 of the modified cysteines and also at 2 lysine residues. The pattern of cleavage in the latter case can be interpreted in terms of the amino acid sequence of the polypeptide.     

Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase

Three cysteine residues, which are completely conserved among alpha-subunits in all nitrile hydratases, are thought to be the ligands of a metal ion in the catalytic center of this enzyme. These cysteine residues (i.e. alpha C102, alpha C105 and alpha C107) in the high-molecular-mass nitrile hydratase (H-NHase) of Rhodococcus rhodochrous J1 were replaced with alanine by site-directed mutagenesis using the R. rhodochrous ATCC12674 host-vector system, and the resultant transformants were investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the cell-free extracts of each mutant transformant revealed that four mutant transformants (i.e. alpha C105A, alpha C107A, alpha C102A/C105A and alpha C105A/C107A) showed predominant alpha- and beta-subunit protein bands with a mobility identical to those of the native H-NHase, while three mutant transformants (i.e. alpha C102A, alpha C102A/C107A and alpha C102A/C105A/C107A) did not produce the corresponding proteins. The purified former four mutant enzymes showed neither enzymatic activity nor the maximum absorption at 410 nm which was detected in the wild type H-NHase. They also did not contain cobalt ions. Based upon these findings, these three cysteine residues were found to be essential for the active expression of H-NHase.     

Conservation of cysteine residues in fungal histidine acid phytases

Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.