Structure/function relationships in OxlT, the oxalate-formate transporter of oxalobacter formigenes. Assignment of transmembrane helix 11 to the translocation pathway

OxlT, the oxalate:formate antiporter of Oxalobacter formigenes, has a lone charged residue, lysine 355 (Lys-355), at the center of transmembrane helix 11 (TM11). Because Lys-355 is the only charged residue in the hydrophobic sector, we tested the hypothesis that lysine 355 contributes to the binding site for the anionic substrate, oxalate. This idea was supported by mutational analysis, which showed that of five variants studied (Lys-355 --> Cys, Gly, Gln, Arg, or Thr), residual function was found for only the K355R derivative, in which catalytic efficiency had fallen 2,600-fold. Further insight came from a study of TM11 single-cysteine mutants, using the impermeant, thiol-specific reagents, carboxyethyl methanethiosulfonate and ethyltrimethylammonium methanethiosulfonate. Of the five reactive positions identified in TM11, four were at the cytoplasmic or periplasmic ends of TM11 (S344C and A345C, and G366C and A370C, respectively), whereas the fifth was at the center of the helix (S359C). Added study with carboxyethyl methanethiosulfonate and ethylsulfonate methylthiosulfonate showed that the attack on S359C could be blocked by the presence of the substrate, oxalate, and that protection could be predicted quantitatively by a kinetic model in which S359C is accessible only in the unliganded form of OxlT. Parallel study showed that the proteoliposomes used in such work contained OxlT of right side-out and inside-out orientations in about equal amounts. Accordingly, full inhibition of S359C by the impermeable methanethiosulfonate-linked probes must reflect an approach from both the cytosolic and periplasmic surfaces of the protein. This, coupled with the finding of substrate protection, leads us to conclude that S359C lies on the translocation pathway through OxlT. Since position 359 and 355 lie on the same helical face, we suggest that Lys-355 also lies on the translocation pathway, consistent with the idea that the essential nature of Lys-355 reflects its role in binding the anionic substrate, oxalate.     

Helix proximity in OxlT, the oalate:formate antiporter of oxalobacter formigenes. Cross-linking between TM2 and TM11.

Experiments were designed to evaluate the proximity of transmembrane helices two (TM2) and eleven (TM11) in the tertiary structure of OxlT, the oxalate:formate exchange transporter of Oxalobacter formigenes. A tandem duplication of the Factor Xa protease cleavage site (IEGRIEGR) was inserted into the central cytoplasmic loop of an OxlT cysteine-less derivative in which an endogenous cleavage site had been eliminated by mutagenesis (R248Q). Using this host, double cysteine derivatives were constructed so as to pair one of seventeen positions in TM2 with one of four positions in TM11. Following treatment of membrane vesicles with Cu(II)(1,10-phenanthroline)(3), molecular iodine, or N,N'-o-phenylenedimaleimide, samples were exposed to Factor Xa, and disulfide bond formation was assessed after SDS-polyacrylamide gel electrophoresis by staining with antibody directed against the OxlT C terminus. In the absence of disulfide bond formation, exposure to Factor Xa revealed the expected C-terminal 22-kDa fragment, a result unaffected by the presence of reductant. By contrast, after disulfide formation, OxlT mobility remained at 35 kDa, and appearance of the 22-kDa fragment required addition of 200 mm dithiothreitol prior to electrophoresis. The four TM11 positions chosen for cysteine substitution lie on a helical face known to interact with substrate. Similarly, TM2 positions supporting disulfide trapping were also confined to a single helical face. We conclude that TM2 and TM11 are in close juxtaposition to one another in the tertiary structure of OxlT.     

Cysteine scanning mutagenesis of TM5 reveals conformational changes in OxlT, the oxalate transporter of Oxalobacter formigenes

We constructed a single-cysteine panel encompassing TM5 of the oxalate transporter, OxlT. The 25 positions encompassed by TM5 were largely tolerant of mutagenesis, and functional product was recovered for 21 of the derived variants. For these derivatives, thiol-directed MTS-linked agents (MTSEA, MTSCE, and MTSES) were used as probes of transporter function, yielding 11 mutants that responded to probe treatment, as indicated by effects on oxalate transport. Further study identified three biochemical phenotypes among these responders. Group 1 included seven mutants, exemplified by G151C, displaying substrate protection against probe inhibition. Group 2 was comprised of a single mutant, P156C, which had unexpected behavior. In this case, we observed increased activity if weak acid/base or neutral probes were used, while exposure to probes introducing a fixed charge led to decreased function. In both instances, the presence of substrate prevented the observed response. Group 3 contained three mutants (e.g., S143C) in which probe sensitivity was increased by the presence of substrate. The finding of substrate-protectable probe modification in groups 1 and 2 suggests that TM5 lies on the permeation pathway, as do its structural counterparts, TM2, TM8, and TM11. In addition, we speculate that substrate binding facilitates TM5 conformational changes that allow new regions to become accessible to MTS-linked probes (group 3). These biochemical data are consistent with the recently developed OxlT homology model.     

Analysis of substrate-binding elements in OxlT, the oxalate:formate antiporter of Oxalobacter formigenes

An OxlT homology model suggests R272 and K355 in transmembrane helices 8 and 11, respectively, are critical to OxlT-mediated transport. We offer positive evidence supporting this idea by studying OxlT function after cysteine residues were separately introduced at these positions. Without further treatment, both mutant proteins had a null phenotype when they were reconstituted into proteoliposomes. By contrast, significant recovery of function occurred when proteoliposomes were treated with MTSEA (methanethiosulfonate ethylamine), a thiol-specific reagent that implants a positively charged amino group. In each case, there was a 2-fold increase in the Michaelis constant (K(M)) for oxalate self-exchange (from 80 to 160 microM), along with a 5-fold (K355C) or 100-fold (R272C) reduction in V(max) compared to that of the cysteine-less parental protein. Analysis by MALDI-TOF confirmed that MTSEA introduced the desired modification. We also examined substrate selectivity for the treated derivatives. While oxalate remained the preferred substrate, there was a shift in preference among other substrates so that the normal rank order (oxalate > malonate > formate) was altered to favor smaller substrates (oxalate > formate > malonate). This shift is consistent with the idea that the substrate-binding site is reduced in size via introduction of the SCH(2)CH(2)NH(3)(+) adduct, which generates a side chain that is approximately 1.85 A longer than that of lysine or arginine. These findings lead us to conclude that R272 and K355 are essential components of the OxlT substrate-binding site.     

Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling

The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His(9)-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+ linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.     

Analysis of transmembrane domain 2 of rat serotonin transporter by cysteine scanning mutagenesis

The second transmembrane domain (TM2) of neurotransmitter transporters has been invoked to control oligomerization and surface expression. This transmembrane domain lies between TM1 and TM3, which have both been proposed to contain residues that contribute to the substrate binding site. Rat serotonin transporter (SERT) TM2 was investigated by cysteine scanning mutagenesis. Six mutants in which cysteine replaced an endogenous TM2 residue had low transport activity, and two were inactive. Most of the reduction in transport activity was due to decreased surface expression. In contrast, M124C and G128C showed increased activity and surface expression. Random mutagenesis at positions 124 and 128 revealed that hydrophobic residues at these positions also increased activity. When modeled as an alpha-helix, positions where mutation to cysteine strongly affects expression levels clustered on the face of TM2 surrounding the leucine heptad repeat conserved within this transporter family. 2-(Aminoethyl)-methanethiosulfonate hydrobromide (MTSEA)-biotin labeled A116C and Y136C but not F117C, M135C, or Y134C, suggesting that these residues may delimit the transmembrane domain. None of the cysteine substitution mutants from 117 through 135 were sensitive to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) or MTSEA. However, treatment with MTSEA increased 5-hydroxytryptamine transport by A116C. Activation of A116C by MTSEA was observed only in mutants containing Cys to Ile mutation at position 357, suggesting that modification of Cys-116 activated transport by compensating for a disruption in transport in response to Cys-357 replacement. The reactivity of A116C toward MTSEA was substantially increased in the presence of substrates but not inhibitors. This increase required Na+ and Cl-, and was likely to result from conformational changes during the transport process.     

A glutamine to glutamate mutation at position 170 (Q170E) in the rabbit Na+/glucose cotransporter, rSGLT1, enhances binding affinity for Na+

Using cysteine mutagenesis and chemical modification by methanethiosulfonate derivatives, it was demonstrated that the external putative loop, joining transmembrane segments (TM's) IV-V of rabbit Na+/glucose cotransporter, rSGLT1, forms part of a Na+ binding and voltage sensing domain. Within this region, exposure to cationic (2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) inhibited F163C, A166C, and L173C, but anionic sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) had no effect. Unexpectedly, MTSEA had no effect on Q170C; however, MTSES profoundly altered Q170C charge transfer and turnover, leaving Na+ and sugar binding affinity unchanged, but mutation of glutamine to anionic glutamate (Q170E) shifted V(0.5) to positive potentials, suggesting enhanced Na+ affinity. To clarify the role of glutamine 170 in Na+ interaction, we embarked on a more detailed investigation of Q170E using the two-microelectrode voltage clamping in Xenopus oocytes. Compared to wild-type (wt) rSGLT1, Q170E exhibits (i) a 2-fold decrease in methyl alpha-D-glucopyranoside affinity (-150 to -90 mV), (ii) a 5-fold increase in Na+ affinity (-150 to -100 mV) with less voltage dependency, (iii) reduced Na+ leak, and (iv) two transient current decay constants (tau(fast), tau(slow)) compared to three (tau(fast), tau(medium), tau(slow)) for wt, and computer simulation of Q170E pre-steady-state currents with a four-state kinetic model yields parameters similar to wt SGLT1, except for a reduced Na+ debinding rate constant compared to wt. Taken together, the data strengthen the conclusion that residue 170 lies in the Na+ pathway and provide the first evidence that it participates in determining Na+ binding.     

Cysteine Scanning of CFTR’s First Transmembrane Segment Reveals Its Plausible Roles in Gating and Permeation

Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR’s TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR’s TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR’s gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.     

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: N-ethylmaleimide-sensitive face of helix II

Cys-scanning mutagenesis of helix II in the lactose permease of Escherichia coli [Frillingos, S., Sun, J. et al. (1997) Biochemistry 36, 269-273] indicates that one face contains positions where Cys replacement or Cys replacement followed by treatment with N-ethylmaleimide (NEM) significantly inactivates the protein. In this study, site-directed sulfhydryl modification is utilized in situ to study this face of helix II. [(14)C]NEM labeling of 13 single-Cys mutants, including the nine NEM-sensitive Cys replacements, in right-side-out membrane vesicles is examined. Permease mutants with a single-Cys residue in place of Gly46, Phe49, Gln60, Ser67, or Leu70 are alkylated by NEM at 25 degrees C in 10 min, and mutants with Cys in place of Thr45 and Ser53 are labeled only in the presence of ligand, while mutants with Cys in place of Ile52, Ser56, Leu57, Leu62, Phe63, or Leu65 do not react. Binding of substrate leads to a marked increase in labeling of Cys residues at positions 45, 49, or 53 in the periplasmic half of helix II and a slight decrease in labeling of Cys residues at positions 60 or 67 in the cytoplasmic half. Labeling studies with methanethiosulfonate ethylsulfonate (MTSES) show that positions 45 and 53 are accessible to solvent in the presence of ligand only, while positions 46, 49, 67, and 70 are accessible to solvent in the absence or presence of ligand. Position 60 is also exposed to solvent, and substrate binding causes a decrease in solvent accessibility. The findings demonstrate that the NEM-sensitive face of helix II participates in ligand-induced conformational changes. Remarkably, this membrane-spanning face is accessible to the aqueous phase from the periplasmic side of the membrane. In the following paper in this issue [Venkatesan, P., Hu, Y., and Kaback, H. R. (2000) Biochemistry 39, 10656-10661], the approach is applied to helix X.     

Site-directed sulfhydryl labeling of helix IX in the lactose permease of Escherichia coli

Site-directed sulfhydryl modification of transmembrane helix IX in the lactose permease of Escherichia coli was studied in right-side-out membrane vesicles with the thiol-specific reagents N-[(14)C]ethylmaleimide (NEM) and methanethiosulfonate ethylsulfonate (MTSES) which are permeant and impermeant, respectively. Out of approximately 20 mutants with a single Cys residue at each position in the helix, only five mutants label with NEM. (i) Cys residues at positions 291, 308, and 310 label at 25 degrees C, and binding of substrate has no effect. (ii) Cys residues at positions 295 and 298 label only in the presence of substrate. NEM labeling at 0 degrees C indicates that alkylation of Cys residues at positions 295 and 308 is dependent on the thermal motion of the protein. In contrast, temperature has little effect on labeling of Cys residues at positions 291, 298, and 310. Interestingly, pretreatment with MTSES blocks NEM labeling of all the mutants. The findings demonstrate that the face of helix IX on which Arg302 is located is involved in ligand-induced conformational changes and accessible to water from the periplasmic surface of the membrane. Since Arg302 facilitates deprotonation of Glu325 (helix X) during turnover [Sahin-Tóth, M., and Kaback, H. R. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6068-6073], the findings are consistent with the idea that this face of helix IX may comprise part of the H(+) translocation pathway.     

Measurement of the substrate dissociation constant of a solubilized membrane carrier. Substrate stabilization of OxlT, the anion exchange protein of Oxalobacter formigenes.

OxlT, a secondary carrier found in Oxalobacter formigenes, mediates the exchange of divalent oxalate and monovalent formate. Because OxlT has an unusually high turnover number (greater than or equal to 1000/s), and because formate, one its substrates, shows high passive membrane permeability as formic acid, it has been difficult to obtain information on protein-substrate interactions using traditional methods in membrane biology. For this reason, we devised a new way to measure substrate dissociation constants. Detergent-solubilized material was exposed to inactivating temperatures in the absence or presence of OxlT substrates, and periodic reconstitution was used to monitor the kinetics of thermal decay. The data were consistent with a simple scheme in which only unliganded OxlT was temperature-sensitive; this premise, along with the assumption of equilibrium between liganded and unliganded species, allowed calculation of substrate dissociation constants for oxalate (18 +/- 3 microM), malonate (1.2 +/- 0.2 mM), and formate (3.1 +/- 0.6 mM). Further analysis revealed that substrate binding energy contributed at least 3.5 kcal/mol to stabilization of solubilized OxlT. Accordingly, we suggest that substrate binding energy is directly involved in driving protein structure reorganization during membrane transport. This new approach to analyzing protein-substrate interactions may have wider application in the study of membrane carriers.     

Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that transmembrane regions I and II, but not III, are structural components of the aqueous translocation channel

The contribution of transmembrane regions I, II, and III of the Rickettsia prowazekii ATP/ADP translocase to the structure of the putative water-filled ATP translocation channel was evaluated from the accessibility of hydrophilic, thiol-reactive, methanethiosulfonate reagents to a library of 68 independent cysteine-substitution mutants heterologously expressed in Escherichia coli. The MTS reagents used were MTSES (negatively charged) and MTSET and MTSEA (both positively charged). Mutants F036C, Y042C, and R046C (TM I), K066C and P072C (TM II), and F101C, F105C, F108C, Y113C, and P114C (TM III) had no assayable transport activity, indicating that cysteine substitution at these positions may not be tolerated. All three MTS reagents inhibit the transport of ATP in mutants of TM I (L039C, S043C, S047C, I048C) and TM II (S061C, S063C, T067C, I069C, V070C, A074C). Further, these residues appear to cluster along a single face of the transmembrane domain. Preexposure of MTS-reactive mutants S047C (TM I) and T067C (TM II) to high levels of ATP resulted in protection from MTS-mediated inhibition. This indicated that both TM I and TM II make major contributions to the structure of an aqueous ATP translocation pathway. Finally, on the basis of the lack of accessibility of charged MTS reagents to the thiol groups in mutants of TM III, it appears that TM III is not exposed to the ATP translocation channel. Cysteine substitution of residues constituting a highly conserved "phenylalanine face" in TM III resulted in ablation of ATP transport activity. Further, substituting these phenylalanine residues for either isoleucine or tyrosine also resulted in much lower transport activity, indicating that some property of phenylalanine at these positions that is not shared by cysteine, isoleucine, or tyrosine is critical to translocase activity.     

The substrates of the gamma-aminobutyric acid transporter GAT-1 induce structural rearrangements around the interface of transmembrane domains 1 and 6

The sodium- and chloride-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 is essential for efficient synaptic transmission by this neurotransmitter. GAT-1 is the first cloned member of the neurotransmitter-sodium-symporter family. Here we address the idea that during transport the extracellular halves of transmembrane domains (TM) 1 and 6, TM 1b/TM 6a, move relative to the binding pocket. Therefore, we have probed the aqueous accessibility of TM 6a and its proximity to TM 1b in the presence and absence of its substrates. Cysteines were introduced, one by one, at all TM 6a positions. In several mutants, transport activity was inhibited by the impermeant sulfhydryl reagent (2-trimethylammonium)methanethiosulfonate, whereas wild type GAT-1 was basically insensitive. This inhibition was potentiated by sodium, whereas GABA was protective. Moreover, we used paired cysteine mutagenesis in conjunction with treatments with copper(II)(1,10-phenanthroline)(3) (CuPh). CuPh did not affect the activity of wild type GAT-1 but potently inhibited transport by the TM 6a mutant D287C. Such inhibition was not observed with D287C/C74A, indicating that Asp-287 is close to Cys-74 of TM 1b. Inhibition of transport of D287C by CuPh, but not by (2-trimethylammonium)methanethiosulfonate, was potentiated when sodium and GABA were both removed. Thus, the degree of inhibition by CuPh is not a simple function of the accessibility of the individual cysteines but also involves structural rearrangements around the TM 1b/TM 6a interface.     

Cysteine-scanning mutagenesis of the fifth external loop of serotonin transporter

External loop 5 (EL5) of serotonin transporter was analyzed by mutating each of the residues from Thr-480 to Ala-511, one at a time, with cysteine. Cysteine was well-tolerated at most positions, although G485C, Y495C, and E508C had low transport activities. Replacement with cysteine rendered mutants G484C-P499C sensitive to partial or complete inactivation by [2-(trimethylammonium)ethyl] methanethiosulfonate and (2-sulfonatoethyl) methanethiosulfonate. Within this sensitive region, the rates of reaction varied by over 2 orders of magnitude. Rates of inactivation were not significantly affected by removal of Na(+) or by addition of cocaine or serotonin. These results suggest that modification of EL5 interferes with the transport process but is not sensitive to substrate and ion binding.     

Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore

Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.     

Cytosolic half of transmembrane domain IV of the human bile acid transporter hASBT (SLC10A2) forms part of the substrate translocation pathway

We report the involvement of transmembrane domain 4 (TM4) of hASBT in forming the putative translocation pathway, using cysteine-scanning mutagenesis in conjunction with solvent-accessibility studies using the membrane-impermeant, sulfhydryl-specific methanethiosulfonate reagents. We individually mutated each of the 21 amino acids in TM4 to cysteine on a fully functional, MTS-resistant C270A-hASBT template. The single-cysteine mutants were expressed in COS-1 cells, and their cell surface expression levels, transport activities [uptake of the prototypical hASBT substrate taurocholic acid (TCA)], and sensitivities to MTS exposure were determined. Only P161 lacked cell-surface expression. Overall, cysteine replacement was tolerated at charged and polar residues, except for mutants I160C, Y162C, I165C, and G179C (相似文献   

Functional role of critical stripe residues in transmembrane span 7 of the serotonin transporter. Effects of Na+, Li+, and methanethiosulfonate reagents

Mutations at critical residue positions in transmembrane span 7 (TM7) of the serotonin transporter affect the Na(+) dependence of transport. It was possible that these residues, which form a stripe along one side of the predicted alpha-helix, formed part of a water-filled pore for Na(+). We tested whether cysteine substitutions in TM7 were accessible to hydrophilic, membrane-impermeant methanethiosulfonate (MTS) reagents. Although all five cysteine-containing mutants tested were sensitive to these reagents, noncysteine control mutants at the same positions were in most cases equally sensitive. In all cases, MTS sensitivity could be traced to changes in accessibility of a native cysteine residue in extracellular loop 1, Cys-109. Moreover, none of the TM7 cysteines reacted with the biotinylating reagent MTSEA-biotin when tested in the C109A background. It is thus unlikely that the critical stripe forms part of a water-filled pore. Instead, studies of the ion dependence of the reaction between Cys-109 and MTS reagents lead to the conclusion that TM7 is involved in propagating conformational changes caused by ion binding, perhaps as part of the translocation mechanism. The critical stripe residues on TM7 probably represent a close contact region between TM7 and one or more other TMs in the transporter's three-dimensional structure.     

Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating

Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single "gate" within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)(2)(-) ions was reduced when ATP concentration was reduced from 1mM to 10μM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)(2)(-) was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.     

Structure and transport mechanism of the bacterial oxalate transporter OxlT

Membrane proteins that belong to the major facilitator superfamily (MFS) are found in organisms across the evolutionary spectrum and mediate the transport of a variety of substrates ranging from small metabolites to neurotransmitters. The oxalate transporter (OxlT) is a representative MFS protein, and exchanges formate for oxalate across the cytoplasmic membrane of the organism Oxalobacter formigenes. Here, we present a structural model for the protein conformational changes that occur during oxalate transport by combining a three-dimensional map of the oxalate-bound, "closed" state of OxlT at 6.5 A determined by cryo-electron microscopy with a model of the "open" state of OxlT based on the atomic structures of the related transporters, glycerol-3-phosphate transporter (GlpT) and lactose permease (LacY). We demonstrate that the principal structural change associated with substrate transport is a concerted rocking movement of the two structurally similar halves of the protein relative to each other. Our structural model places two positively charged residues, Arg-272 and Lys-355 in the central cavity, suggesting that electrostatic interactions between these residues and the oxalate anion is a key step in generating the conformational change between the open and closed states of the transporter.     

The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium

P-Glycoprotein (P-gp) is an ATP-dependent drug pump that transports a broad range of compounds out of the cell. Cross-linking studies have shown that the drug-binding pocket is at the interface between the transmembrane (TM) domains and can simultaneously bind two different drug substrates. Here, we determined whether cysteine residues within the drug-binding pocket were accessible to the aqueous medium. Cysteine mutants were tested for their reactivity with the charged thiol-reactive compounds sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) and [2-(trimethylammonium)ethyl)]methanethiosulfonate (MTSET). Residue Ile-306(TM5) is close to the verapamil-binding site. It was changed to cysteine, reacted with MTSES or MTSET, and assayed for verapamil-stimulated ATPase activity. Reaction of mutant I306C(TM5) with either compound reduced its affinity for verapamil. We confirmed that the reduced affinity for verapamil was indeed due to introduction of a charge at position 306 by demonstrating that similar effects were observed when Ile-306 was replaced with arginine or glutamic acid. Mutant I306R showed a 50-fold reduction in affinity for verapamil and very little change in the affinity for rhodamine B or colchicine. MTSES or MTSET modification also affected the cross-linking pattern between pairs of cysteines in the drug-binding pocket. For example, both MTSES and MTSET inhibited cross-linking between I306C(TM5) and I868C(TM10). Inhibition was enhanced by ATP hydrolysis. By contrast, cross-linking of cysteine residues located outside the drug-binding pocket (such as G300C(TM5)/F770C(TM8)) was not affected by MTSES or MTSET. These results indicate that the drug-binding pocket is accessible to water.