Cysteine substitutions define etomidate binding and gating linkages in the α-M1 domain of γ-aminobutyric acid type A (GABAA) receptors.

Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABAA receptors. Photoreactive etomidate derivatives labeled αMet-236 in transmembrane domain M1, which structural models locate in the ß+/α- subunit interface. Other nearby residues may also contribute to etomidate binding and/or transduction through rearrangement of the site. In human α1ß2γ2L GABAA receptors, we applied the substituted cysteine accessibility method to α1-M1 domain residues extending from α1Gln-229 to α1Gln-242. We used electrophysiology to characterize each mutant's sensitivity to GABA and etomidate. We also measured rates of sulfhydryl modification by p-chloromercuribenzenesulfonate (pCMBS) with and without GABA and tested if etomidate blocks modification of pCMBS-accessible cysteines. Cys substitutions in the outer α1-M1 domain impaired GABA activation and variably affected etomidate sensitivity. In seven of eight residues where pCMBS modification was evident, rates of modification were accelerated by GABA co-application, indicating that channel activation increases water and/or pCMBS access. Etomidate reduced the rate of modification for cysteine substitutions at α1Met-236, α1Leu-232 and α1Thr-237. We infer that these residues, predicted to face ß2-M3 or M2 domains, contribute to etomidate binding. Thus, etomidate interacts with a short segment of the outer α1-M1 helix within a subdomain that undergoes significant structural rearrangement during channel gating. Our results are consistent with in silico docking calculations in a homology model that orient the long axis of etomidate approximately orthogonal to the transmembrane axis.

Structure of the M2 transmembrane segment of GLIC, a prokaryotic Cys loop receptor homologue from Gloeobacter violaceus, probed by substituted cysteine accessibility.

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11'), I235C (12'), V241C (18'), T243C (20'), L245C (22'), and Y250C (27'), which were not functional, except for 12'. Currents for P246C (23') and K247C (24') were only significantly altered at pH 5.0. The reaction rates were all >1000 m(-1) s(-1). The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10' to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.

Inhibition of human NTPDase 2 by modification of an intramembrane cysteine by p-chloromercuriphenylsulfonate and oxidative cross-linking of the transmembrane domains.

Human NTPDase 2 is a cell surface integral membrane glycoprotein that is anchored to the membranes by two transmembrane domains while the bulk of the protein containing the active site faces the extracellular milieu. It contains 10 conserved cysteine residues in the extracellular domain that are involved in disulfide bond formation and one free cysteine residue, C26, which is located in the N-terminal transmembrane domain. The human NTPDase 2 activity is inactivated by membrane perturbation that disrupts interaction of the transmembrane domains and is inhibited by p-chloromercuriphenylsulfonate (pCMPS), a sulfhydryl reagent. In this report, we show that C26 is the target of pCMPS modification, since a mutant in which C26 was replaced with a serine was no longer inhibited by pCMPS. Mutants in which cysteine residues are placed in the C-terminal transmembrane domain near the extracellular surface were still modified by pCMPS, but the degree of inhibition of their ATPase activity was lower than that of the wild-type enzyme. Thus, loss of the ATPase activity of human NTPDase 2 in the presence of pCMPS probably results from the disturbance of both transmembrane domain interaction and its active site. Inhibition of human NTPDase 2 activity by pCMPS and membrane perturbation is attenuated when the enzyme is cross-linked by glutaraldehyde. On the other hand, NTPDase 2 dimers formed from oxidative cross-linking of the wild-type enzyme and mutants containing a single cysteine residue in the C-terminal transmembrane domain displayed reduced ATPase activity. A similar reduction in activity was also obtained upon intramolecular disulfide formation in mutants that contain a cysteine residue in each of the two transmembrane domains. These results indicate that the mobility of the transmembrane helices is necessary for maximal catalysis.

A phosphatase inhibitor enhances the DNase I sensitivity of active chromatin.

Although it is well-known that active domains of chromatin have elevated DNase I sensitivity, it can be difficult to observe preferential sensitivity in many cell types. We show that the DNase I sensitivity of active chromatin is enhanced some 10-fold by treating nuclei with the phosphatase inhibitor p-(chloromercuri)benzenesulfonic acid (CMBS) whereas DNase I sensitivity in inactive domains is only 3-fold higher. We further show that CMBS-enhanced DNase I sensitivity is associated with at least two histone modifications. First, the negatively charged CMBS molecule becomes covalently attached to the thiol groups on histone H3. Second, histone H2A phosphorylation is significantly elevated in treated nuclei. The phosphorylation data along with other results point to the possibility that H2A phosphorylation plays a role in enhancing preferential DNase I sensitivity. Whatever the mechanism, CMBS treatment of nuclei followed by DNase I digestion provides a novel and reproducible assay for probing the chromatin structure of active domains.

Characterization of a novel polypeptide N-acetylgalactosaminyltransferase (dGalNAc-T3) from Drosophila.

Polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases) catalyze the initial reaction of mucin-type O-glycosylation. Here, we report the first biochemical characterization of one of the Drosophila GalNAc-transferases, dGalNAc-T3. This enzyme retains conserved motifs essential for the catalytic activity, but is a novel isozyme in that it has several inserted sequences in its lectin-like domain. Northern hybridization analysis of this isozyme identified a 2.5-kb mRNA in Drosophila larva. Biochemical characterization was carried out using the recombinant soluble dGalNAc-T3 expressed in COS7 cells. dGalNAc-T3, which required Mn2+ for the activity, had a pH optimum ranging from pH 7.5 to 8.5, and glycosylated most effectively at 29-33 degrees C. Its Km for UDP-GalNAc was 10.7 microM, which is as low as that of mammalian isozymes. dGalNAc-T3 glycosylated the peptides containing a sequence of XTPXP or TTAAP most efficiently. The enzyme was irreversibly inhibited by p-chloromercuriphenylsulphonic acid, indicating the presence of essential Cys residues for the activity.

Analysis of transmembrane segment 8 of the GLUT1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility.

The GLUT1 glucose transporter has been proposed to form an aqueous substrate translocation pathway via the clustering of several amphipathic transmembrane helices (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945). The possible role of transmembrane helix 8 in the formation of this permeation pathway was investigated using cysteine-scanning mutagenesis and the membrane-impermeant sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one GLUT1 mutants were created from a fully functional cysteine-less parental GLUT1 molecule by successively changing each residue along transmembrane segment 8 to a cysteine. The mutant proteins were then expressed in Xenopus oocytes, and their membrane concentrations, 2-deoxyglucose uptake activities, and sensitivities to pCMBS were determined. Four positions within helix 8, alanine 309, threonine 310, serine 313, and glycine 314, were accessible to pCMBS as judged by the inhibition of transport activity. All four of these residues are clustered along one face of a putative alpha-helix. These results suggest that transmembrane segment 8 of GLUT1 forms part of the sugar permeation pathway. Updated two-dimensional models for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose binding site of GLUT1 are proposed that are consistent with existing experimental data and homology modeling based on the crystal structures of two bacterial membrane transporters.

Cysteine-scanning mutagenesis of flanking regions at the boundary between external loop I or IV and transmembrane segment II or VII in the GLUT1 glucose transporter.

To investigate local secondary structure of GLUT1, site-directed and cysteine-scanning mutagenesis were employed to probe p-chloromercuribenzenesulfonate sensitivity of flanking regions at the boundary of external loops (ELs) and transmembrane segments (TMs) and to check the compatibility of two alternative membrane topology models with the experimental data. In the Cys-less GLUT1, single serine residues located in external loops adjacent to putative transmembrane segments were replaced with cysteine. Transport activities of the cysteine-replacement mutants were comparable to that of the nonmutated Cys-less GLUT1. Only the cysteine residues inserted into the first or fourth EL contributed to transport inhibition by p-chloromercuribenzenesulfonate (pCMBS). Dependent on the pCMBS sensitivity of these residues, cysteine-scanning mutagenesis of flanking regions was performed, including EL I-TM II and TM VII-EL IV, respectively. Of the 27 amino acids changed, the majority of cysteine-substitution mutants displayed transport activities comparable to that of Cys-less GLUT1. Irreplaceable amino acids were Phe-72, Gly-286, Asp-288, Tyr-292, and Tyr-293. The pCMBS sensitivity of loop residues decreased when the distance between inserted thiol groups and the putative transmembrane limit increased. The mutants T62C, T63C, T295C, and I297C even exhibited transport stimulation after pCMBS treatment. Regarding putative membrane-harbored residues, a few thiol groups were involved in pCMBS-induced transport inhibition. Drawn on a helix wheel, these pCMBS-sensitive cysteine residues lie on the same facial half of the helix, shown for TM II and TM VII. With respect to EL-TM boundaries, the experimental data are consistent with the local secondary structure predicted from hydropathy profiles. Conversely, certain data obtained by pCMBS-sensitivity scanning are not consistent with either of the two recently published alternative GLUT1 topology models.

Identification of residues in the translocation pathway of EmrE, a multidrug antiporter from Escherichia coli.

EmrE is a small, 12-kDa, highly polyspecific antiporter, which exchanges hydrogen ions with aromatic cations such as methyl viologen. EmrE-mediated transport is inhibited by the sulfhydryl-reactive reagent 4-(chloromercuri)benzoic acid (PCMB) but not by a variety of other sulfhydryl reagents. This differential effect is due to the fact that the organic mercurial is a substrate of the transporter and can reach domains otherwise inaccessible to the different reagents. To find out which of the three cysteine residues in EmrE is reacting with PCMB, each was replaced with serine and it was shown that none of them is essential for transport activity. A protein completely devoid of Cys residues (CL) is also capable of substrate accumulation albeit at a slower rate. Mutated proteins in which only one of the native cysteines was left whereas the other changed to serine were also constructed. The use of these proteins demonstrated that two of the three Cys in EmrE, Cys-41 and Cys-95, but not Cys-39, react with PCMB. A related mercurial, 4-(chloromercuri)benzenesulfonic acid (PCMBS), is only a very poor inhibitor, probably because of the negative charge it bears. PCMBS reacts with EmrE in an asymmetric and unique way. It reacts with the mutant bearing a single Cys residue in position 95 (CL-C95) only when the reagent is present in the outside face of the membrane and with the mutant CL-C41 only when allowed to permeate to the cell interior; as expected, it does not react with the mutant protein bearing a single Cys at position 39 (CL-C39). It is concluded that PCMB permeates through the substrate pathway of EmrE and covalently reacts with the two exposed residues, Cys-95 and Cys-41, but not with Cys-39, located on the opposite face of the helix relative to residue 41. In addition, because of the asymmetric reactivity to PCMBS, an inhibitor that does not permeate through the protein, it is concluded that Cys-41 is closer to the cytoplasmic face than Cys-95. The results demonstrate the existence of a domain accessible only to substrates and provide a unique tool for studying the substrate permeation pathway of an ion-coupled transporter.

Cysteine-less glycosylphosphatidylinositol-specific phospholipase C is inhibited competitively by a thiol reagent: evidence for glyco-mimicry by p-chloromercuriphenylsulphonate.

Glycosylphosphatidylinositol (GPI)-specific phospholipases are highly valuable for studying the structure and function of GPIs. GPI-specific phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus are the most widely studied of this class of phospholipases C. Inhibition of protein activity by thiol reagents is indicative of the participation of cysteine residues in biochemical events. The thiol reagent p-chloromercuriphenylsulphonate (pCMPS) inhibits T. brucei GPI-PLC, which has eight cysteine residues. Surprisingly, we found that the activity of B. cereus PI-PLC is also blocked by pCMPS, although the protein does not contain cysteine residues. Inhibition of B. cereus PI-PLC was reversed when pCMPS was size-separated from a preformed pCMPS.PI-PLC complex. In contrast, no activity was recovered when T. brucei GPI-PLC was subjected to a similar protocol. Equimolar beta-mercaptoethanol (beta-ME) reversed the inhibition of PI-PLC activity in a pCMPS.PI-PLC complex. For T. brucei GPI-PLC, however, ultrafiltration of the pCMPS.GI-PLC complex and addition of a large excess of beta-ME was necessary for partial recovery of enzyme activity. Thus T. brucei GPI-PLC is susceptible to inactivation by covalent modification with pCMPS, whereas PI-PLC is not. Kinetic analysis indicated that pCMPS was a competitive inhibitor of PI-PLC when a GPI was a substrate. Curiously, with phosphatidylinositol as substrate, inhibition was no longer competitive. These data suggest that pCMPS is a glyco-mimetic that occupies the glycan binding site of PI-PLC, from where, depending on the substrate, it inhibits catalysis allosterically or competitively.

Defining the propofol binding site location on the GABAA receptor.

The GABAA receptor is a target of many general anesthetics. The low affinity of general anesthetics has complicated the search for the location of anesthetic binding sites. Attention has focused on two pairs of residues near the extracellular ends of the M2 and M3 membrane-spanning segments, alpha1Ser270/beta2Asn265 (15' M2) and alpha1Ala291/beta2Met286 (M3). In the 4-A resolution acetylcholine receptor structure, the aligned positions are separated by approximately 10 A. To determine whether these residues are part of a binding site for propofol, an intravenous anesthetic, we probed propofol's ability to protect cysteines substituted for these residues from modification by the sulfhydryl-specific reagent p-chloromercuribenzenesulfonate (pCMBS-). pCMBS- reacted with cysteines substituted at the four positions in the absence and presence of GABA. Because propofol binding induces conformational change in the GABAAreceptor, we needed to establish a reference state of the receptor to compare reaction rates in the absence and presence of propofol. We compared reaction rates in the presence of GABA with those in the presence of propofol +GABA. The GABA concentration was reduced to give a similar fraction of the maximal GABA current in both conditions. Propofol protected, in a concentration-dependent manner, the cysteine substituted for beta2Met286 from reaction with pCMBS-. Propofol did not protect the cysteine substituted for the aligned alpha1 subunit position or the 15' M2 segment Cys mutants in either subunit. We infer that propofol may bind near the extracellular end of the betasubunit M3 segment.

Cysteine-scanning mutagenesis of transmembrane segment 1 of glucose transporter GLUT1: extracellular accessibility of helix positions.

Transmembrane segment 1 of the cysteine-less GLUT1 glucose transporter was subjected to cysteine-scanning mutagenesis. The majority of single-cysteine mutants were functional transporters, as assessed by 2-deoxy-d-glucose uptake or 3-O-methyl-d-glucose transport. Substitution of cysteine for Leu-21, Gly-22, Ser-23, Gln-25, and Gly-27, however, led to uptake rates that were less than 10% of that of the nonmutated cysteine-less GLUT1. NEM, a membrane-permeable agent, was used to identify positions that are sensitive to transport alteration by sulfhydryl reagents, whereas uptake modification by the membrane-impermeant pCMBS indicated accessibility to water-soluble solutes from the external cell environment. Twelve of the 21 single-cysteine mutants were significantly (p < 0.01) affected by NEM, and on the basis of this sensitivity, four positions were identified by pCMBS to form a water-accessible surface within helix 1. The pCMBS-sensitive positions are localized at the exofacial C-terminal end along a circumference of the helix.