Analysis of transmembrane segment 10 of the Glut1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility.

The Glut1 glucose transporter has been proposed to form an aqueous sugar translocation pathway through the lipid bilayer via the clustering of several 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 participation of transmembrane helix 10 in the formation of this putative aqueous tunnel was tested using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteine-less, parental Glut1 molecule by changing each residue within putative transmembrane segment 10 to cysteine. Each mutant was then expressed in Xenopus oocytes, and its plasma membrane content, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. Helix 10 exhibited a highly distinctive reaction profile to scanning mutagenesis whereby cysteine substitution at residues within the cytoplasmic N-terminal half of the helix tended to increase specific transport activity, whereas substitution at residues within the exoplasmic C-terminal half of the helix tended to decrease specific transport activity. Four residues within helix 10 were clearly accessible to pCMBS as judged by inhibition or stimulation of transport activity. All four of these residues were clustered along one face of a putative alpha-helix. These results combined with previously published data suggest that transmembrane segment 10 of Glut1 forms part of the sugar permeation pathway. Two-dimensional models for the conformation of the 12 transmembrane helices and the exofacial glucose-binding site of Glut1 are proposed that are consistent with existing experimental data.     

Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter

The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning alpha-helices based upon hydropathy plot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, and 11) are capable of forming amphipathic structures. A model of GLUT1 tertiary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues in transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutants retain measurable transport activity. Over one-half of the cysteine mutants had significantly reduced specific activity relative to the C-less protein. The solvent accessibility and relative orientation of the residues within the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagent p-chloromercuribenzene sulfonate (pCMBS). Cysteine replacement at six positions (Gln(282), Gln(283), Ile(287), Ala(289), Val(290), and Phe(291)), all near the exofacial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. These data demonstrate that the exofacial portion of transmembrane segment 7 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within the glucose permeation pathway.     

Transmembrane segment 5 of the Glut1 glucose transporter is an amphipathic helix that forms part of the sugar permeation pathway

Transmembrane segment 5 of the Glut1 glucose transporter has been proposed to form an amphipathic transmembrane helix that lines the substrate translocation pathway (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). This hypothesis was tested using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteine-less, parental Glut1 molecule by changing each residue within putative transmembrane segment 5 to cysteine. Each mutant was then expressed in Xenopus oocytes and its steady-state protein level, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. All 21 mutants exhibited measurable transport activity, although several of the mutants exhibited reduced activity due to a corresponding reduction in steady-state protein. Six of the amino acid side chains within transmembrane segment 5 were clearly accessible to pCMBS in the external medium, as determined by inhibition of transport activity, and a 7th residue showed inhibition that lacked statistical significance because of the extremely low transport activity of the corresponding mutant. All 7 of these residues were clustered along one face of a putative alpha-helix, proximal to the exoplasmic surface of the plasma membrane. These results comprise the first experimental evidence for the existence of an amphipathic transmembrane alpha-helix in a glucose transporter molecule and strongly suggest that transmembrane segment 5 of Glut1 forms part of the sugar permeation pathway.     

Cysteine scanning mutagenesis of helices 2 and 7 in GLUT1 identifies an exofacial cleft in both transmembrane segments

Cysteine scanning mutagenesis in conjunction with site-directed chemical modification of sulfhydryl groups by p-chloromercuribenzenesulfonate (pCMBS) or N-ethylmaleimide (NEM) was applied to putative transmembrane segments (TM) 2 and 7 of the cysteine-less glucose transporter GLUT1. Valid for both helices, the majority of cysteine substitution mutants functioned as active glucose transporters. The residues F72, G75, G76, G79, and S80 within helix 2 and G286 and N288 within helix 7 were irreplaceable because the mutant transporters displayed transport activities that were lower than 10% of Cys-less GLUT1. The indicated cluster of glycine residues within TM 2 is located on one face of the helix and may provide space for a bulky hydrophobic counterpart interacting with another transmembrane segment or lipid side chains. Characteristic for helix 7, three glutamine residues (Q279, Q282, and Q283) played an important role in transport activity of Cys-less GLUT1 because an individual replacement with cysteine reduced their transport rates by about 80%. ParaCMBS-sensitivity scanning of both transmembrane segments detected several membrane-harbored residues to be accessible to the extracellular aqueous solvent. The pCMBS-reactive sulfhydryl groups were located exclusively in the exofacial half of the plasma membrane and, when presented in a helical model, lie along one side of the helices. Taken together, transmembrane segments 2 and 7 form clefts accessible to the extracellular aqueous solvent. The lining residues are however excluded from interaction with intracellular solutes, as justified by microinjection of pCMBS into the cytoplasm of Xenopus oocytes.     

Cysteine-scanning mutagenesis of transmembrane segment 11 of the GLUT1 facilitative glucose transporter

The glucose permeation pathway within the GLUT1 facilitative glucose transporter is hypothesized to be formed by the juxtaposition of the hydrophilic faces of several transmembrane alpha-helices. The role of transmembrane segment 11 in forming a portion of this central aqueous channel was investigated using cysteine-scanning mutagenesis in conjunction with sulfhydryl-directed chemical modification. Each of the amino acid residues within transmembrane segment 11 were individually mutated to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that all of these mutants retain measurable transport activity. Four of the cysteine mutants (N411, W412, N415, and F422) had significantly reduced specific activity relative to the C-less protein. Specific activity was increased in five of the mutants (A402, A405, V406, F416, and M420). The solvent accessibility and relative orientation of the residues to the glucose permeation pathway were investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl-directed reagent p-chloromercuribenzenesulfonate (pCMBS). Cysteine replacement at five positions (I404, G408, F416, G419, and M420) produced transporters that were inhibited by incubation with extracellular pCMBS. All of these residues cluster along a single face of the alpha-helix within the regions showing altered specific activities. These data demonstrate that the exofacial portion of transmembrane segment 11 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within or near the glucose permeation pathway.     

Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism

A model has been proposed for the exofacial configuration of the Glut1 glucose transporter in which eight transmembrane domains form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 12, predicted to be an outer helix in this hypothetical model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A previously characterized functional cysteine-less Glut1 molecule was used to produce 21 Glut1 point mutants by changing each residue along helix 12 to a cysteine residue. These mutants were then expressed in Xenopus oocytes, and their protein levels, functional activities, and sensitivities to pCMBS were determined. Strikingly, in contrast to all nine other predicted Glut1 transmembrane helices that have been previously examined by this method, none of the 21 helix 12 single-cysteine mutants exhibited significant inhibition of specific transport activity. Also unlike most other Glut1 transmembrane domains in which solvent-accessible residues lie along a single face of the helix, mutations in five consecutive residues predicted to lie close to the exofacial face of the membrane resulted in sensitivity to pCMBS-induced transport inhibition. These results suggest that helix 12 plays a passive stabilizing role in the structure of Glut1 and is not directly involved in the transport mechanism. Additionally, the pCMBS data indicate that the predicted exoplasmic end of helix 12 is completely exposed to the external solvent when the transporter is in its exofacial configuration.     

Analysis of glucose transporter topology and structural dynamics

Homology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6-7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and d-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not D-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by alpha-chymotrypsin, which cuts putative pore-forming amphipathic alpha-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to alpha-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate.     

Transmembrane segment 3 of the Glut1 glucose transporter is an outer helix

A model has been proposed for the structure of the Glut1 glucose transporter based on the results of mutagenesis studies and homology modeling in which eight transmembrane segments form an inner helical bundle surrounded by four outer helices. The role of transmembrane segment 3 in this structural model was investigated using cysteine-scanning mutagenesis in conjunction with 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 helix 3 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. Cysteine substitution at methionine 96 abolished transport activity, whereas substitutions at the other positions resulted in either modest reductions or no significant effect on transport activity. In striking contrast to all other helices that have been examined to date, only one of the 21 helix 3 single-cysteine mutants was inhibited by pCMBS, suggesting that only a small portion of this helix is exposed to the external solvent. This result is consistent with predictions based on our current structural model, in which helix 3 is one of four outer helices that surround the inner helical bundle that comprises the aqueous substrate-binding cavity. An updated two-dimensional model for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose-binding site of Glut1 is presented that is consistent with existing experimental data.     

Transmembrane segment 6 of the Glut1 glucose transporter is an outer helix and contains amino acid side chains essential for transport activity

Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. Cysteine substitutions at Leu(204) and Pro(205) abolished transport activity, whereas substitutions at Ile(192), Pro(196), Gln(200), and Gly(201) resulted in inhibition of activity that ranged from approximately 35 to approximately 80%. Cysteine substitutions at Leu(188), Ser(191), and Leu(199) moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu(188) conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters.     

Relative proximity and orientation of helices 4 and 8 of the GLUT1 glucose transporter

A structure has been proposed for glucose transporter-1 (GLUT1) based upon homology modeling that is consistent with the results of numerous mutagenesis studies (Mueckler, M., and Makepeace, C. (2004) J. Biol. Chem. 279, 10494-10499). To further test and refine this model, the relative orientation and proximity of transmembrane helices 4 and 8 were analyzed by chemical crosslinking of di-cysteine mutants created in a reporter GLUT1 construct. All six native cysteine residues of GLUT1 were changed to either glycine or serine residues by site-directed mutagenesis, resulting in a functional Glut1 construct with Cys mutated to Gly/Ser (C-less). The GLUT1 reporter molecule was engineered from C-less GLUT1 by creating a unique cleavage site for factor Xa protease within the central cytoplasmic loop and by eliminating the site of N-linked glycosylation. Fourteen functional di-cysteine mutants were then created from the C-less reporter construct, each mutant containing a single cysteine residue in helix 4 and one cysteine residue in helix 8. These mutants were expressed in Xenopus oocytes, and the sensitivity of each mutant to intramolecular crosslinking by two homo-bifunctional, thiol-specific crosslinking reagents, bismaleimidehexane and 1,4-phenylenedimaleimide, was ascertained by protease cleavage followed by immunoblot analysis. Four pairs of cysteine residues, Cys(148)/Cys(328), Cys(145)/Cys(328), Cys(148)/Cys(325), and Cys(145)/Cys(325), were observed to be in close enough proximity to be susceptible to crosslinking by one or both reagents. All five of the cysteine residues susceptible to crosslinking are predicted to lie on the same face of helix 4 or 8 and to reside close to the cytoplasmic face of the membrane. These data indicate that the cytoplasmic ends of helices 4 and 8 lie within 6-16 A of one another and that the two helices twist or tilt such that they are further than 16 A apart toward the center and the exoplasmic side of the membrane. An updated model for the clustering of the transmembrane helices of GLUT1 is presented based on these data.     

Cysteine-scanning mutagenesis and substituted cysteine accessibility analysis of transmembrane segment 4 of the Glut1 glucose transporter

A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.     

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.     

Proposed structure of putative glucose channel in GLUT1 facilitative glucose transporter.

A family of structurally related intrinsic membrane proteins (facilitative glucose transporters) catalyzes the movement of glucose across the plasma membrane of animal cells. Evidence indicates that these proteins show a common structural motif where approximately 50% of the mass is embedded in lipid bilayer (transmembrane domain) in 12 alpha-helices (transmembrane helices; TMHs) and accommodates a water-filled channel for substrate passage (glucose channel) whose tertiary structure is currently unknown. Using recent advances in protein structure prediction algorithms we proposed here two three-dimensional structural models for the transmembrane glucose channel of GLUT1 glucose transporter. Our models emphasize the physical dimension and water accessibility of the channel, loop lengths between TMHs, the macrodipole orientation in four-helix bundle motif, and helix packing energy. Our models predict that five TMHs, either TMHs 3, 4, 7, 8, 11 (Model 1) or TMHs 2, 5, 11, 8, 7 (Model 2), line the channel, and the remaining TMHs surround these channel-lining TMHs. We discuss how our models are compatible with the experimental data obtained with this protein, and how they can be used in designing new biochemical and molecular biological experiments in elucidation of the structural basis of this important protein function.     

Mutational analysis of the hexose transporter of Plasmodium falciparum and development of a three-dimensional model

Plasmodium falciparum infection kills more than 1 million children annually. Novel drug targets are urgently being sought as multidrug resistance limits the range of treatment options for this protozoan pathogen. PfHT1, the major hexose transporter of P. falciparum is a promising new target. We report detailed structure-function studies on PfHT1 using site-directed mutagenesis approaches on residues located in helix V (Q169N) and helix VII ((302)SGL --> AGT). Studies with hexose analogues in these mutants have established that hexose recognition and permeation are intimately linked to these helices. A "fructose filter" effect results from the Q169N mutation (abolishing fructose uptake but preserving affinity and transport of glucose, as reported in Woodrow, C. J., Burchmore, R. J. S., and Krishna, S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9931-9936). Associated changes in competition for glucose uptake by C-2, C-3, and C-6 glucose analogues compared with native PfHT1 indicate subtle alterations in substrate interaction in this mutant. The K(m) values for glucose uptake in helix VII mutants are also similar to native PfHT1. Hydrogen bonding to positions C-5 and C-6 in glucose analogues becomes relatively more important in these mutants compared with native PfHT1. To increase understanding of hexose permeation pathways in PfHT1, we have developed the first three-dimensional model for PfHT1. As predicted for GLUT1, the principal mammalian glucose transporter, PfHT1 contains a main and an auxiliary channel. After modeling, the Q169N mutation leads predominantly to local structural changes, including displacement of neighboring helix IV. The (302)SGL position in helix VII lies in the same plane as Gln-169 in helix V but is also adjacent to the main hexose permeation pathway, consistent with results from experiments mutating this triplet motif. Furthermore, there are obvious structural and functional differences between GLUT1 and PfHT1 that can now be explored in detail using the approaches presented here. The development of specific inhibitors for PfHT1 will also be aided by these insights.     

Model of the 3-D structure of the GLUT3 glucose transporter and molecular dynamics simulation of glucose transport

A molecular model of the three-dimensional (3-D) structure of the glucose transport protein, GLUT3, has been derived by homology modeling. The model was built on the basis of structural data from the MscL protein, which is a mechanosensitive ion channel, and general insights from aquaporin (a water permeation pore). Structurally conserved regions were defined by amino acid sequence comparisons, optimum interconnecting loops were selected from the protein databank, and amino (N)- and carboxy (C)-terminal ends of the protein were generated as random coil structures. The model was then subjected to energy minimization and molecular dynamics simulations in the presence of bound substrate (D-glucose). In the proposed structure of GLUT3, the 12 transmembrane (TM) helices form a right-hand barrel with a central hydrophilic pore. The pore is shaped like a funnel with dimensions of approximately 5-6 A by 8 A at its narrowest point. A network of polar and aromatic amino acids line the pore region and may facilitate the movement of glucose along the channel. A putative binding site for inhibitory ligands, such as forskolin and cytochalasin B, was identified on an intracellular aspect of the protein. Molecular dynamics studies showed that changes in the tilt and flexibility of key TM helices may modulate the opening of the pore to effect glucose transport. The proposed structure of GLUT3 may prove useful in guiding future experiments aimed at more precisely defining various functional regions of the transporter and may encourage efforts to develop models of other complex membrane proteins.     

Gating-induced Conformational Rearrangement of the γ-Aminobutyric Acid Type A Receptor β-α Subunit Interface in the Membrane-spanning Domain

GABA(A) receptors mediate fast inhibitory synaptic transmission. The transmembrane ion channel is lined by a ring of five α helices, M2 segments, one from each subunit. An outer ring of helices comprising the alternating M1, M3, and M4 segments from each subunit surrounds the inner ring and forms the interface with the lipid bilayer. The structural rearrangements that follow agonist binding and culminate in opening of the ion pore remain incompletely characterized. Propofol and other intravenous general anesthetics bind at the βM3-αM1 subunit interface. We sought to determine whether this region undergoes conformational changes during GABA activation. We measured the reaction rate of p-chloromercuribenzenesulfonate (pCMBS) with cysteines substituted in the GABA(A) receptor α1M1 and β2M3 segments. In the presence of GABA, the pCMBS reaction rate increased significantly in a cluster of residues in the extracellular third of the α1M1 segment facing the β2M3 segment. Mutation of the β2M2 segment 19' position, R269Q, altered the pCMBS reaction rate with several α1M1 Cys, some only in the resting state and others only in the GABA-activated state. Thus, β2R269 is charged in both states. GABA activation induced disulfide bond formation between β2R269C and α1I228C. The experiments demonstrate that α1M1 moves in relationship to β2M2R269 during gating. Thus, channel gating does not involve rigid body movements of the entire transmembrane domain. Channel gating causes changes in the relative position of transmembrane segments both within a single subunit and relative to the neighboring subunits.     

Structural analysis of synthetic peptide fragments from EmrE,a multidrug resistance protein,in a membrane-mimetic environment

EmrE, a multidrug resistance protein from Escherichia coli, renders the bacterium resistant to a variety of cytotoxic drugs by active translocation out of the cell. The 110-residue sequence of EmrE limits the number of structural possibilities that can be envisioned for this membrane protein. Four helix bundle models have been considered [Yerushalmi, H., Lebendiker, M., and Schuldiner, S. (1996) J. Biol. Chem. 271, 31044-31048]. The validity of EmrE structural models has been probed experimentally by investigations on overlapping peptides (ranging in length from 19 to 27 residues), derived from the sequence of EmrE. The choice of peptides was made to provide sequences of two complete, predicted transmembrane helices (peptides H1 and H3) and two helix-loop-helix motifs (peptides A and B). Peptide (B) also corresponds to a putative hairpin in a speculative beta-barrel model, with the "Pro-Thr-Gly" segment forming a turn. Structure determination in SDS micelles using NMR indicates peptide H1 to be predominantly helical, with helix boundaries in the micellar environment corroborating predicted helical limits. Peptide A adopts a helix-loop-helix structure in SDS micelles, and peptide B was also largely helical in micellar environments. An analogue peptide, C, in which the central "Pro-Thr-Gly" was replaced by "(D)Pro-Gly" displays local turn conformation at the (D)Pro-Gly segment, but neither a continuous helical stretch nor beta-hairpin formation was observed. This study implies that the constraints of membrane and micellar environments largely direct the structure of transmembrane peptides and proteins and study of judiciously selected peptide fragments can prove useful in the structural elucidation of membrane proteins.     

Cholesterol interacts with transmembrane alpha-helices M1, M3, and M4 of the Torpedo nicotinic acetylcholine receptor: photolabeling studies using [3H]Azicholesterol

The photoactivatable sterol probe [3alpha-(3)H]6-Azi-5alpha-cholestan-3beta-ol ([3H]Azicholesterol) was used to identify domains in the Torpedo californica nicotinic acetylcholine receptor (nAChR) that interact with cholesterol. [3H]Azicholesterol partitioned into nAChR-enriched membranes very efficiently (>98%), photoincorporated into nAChR subunits on an equal molar basis, and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine, consistent with photoincorporation at the nAChR lipid-protein interface. Sites of [3H]Azicholesterol incorporation in each nAChR subunit were initially mapped by Staphylococcus aureus V8 protease digestion to two relatively large homologous fragments that contain either the transmembrane segments M1-M2-M3 (e.g., alphaV8-20) or M4 (e.g., alphaV8-10). The distribution of [3H]Azicholesterol labeling between these two fragments (e.g., alphaV8-20, 29%; alphaV8-10, 71%), suggests that the M4 segment has the greatest interaction with membrane cholesterol. Photolabeled amino acid residues in each M4 segment were identified by Edman degradation of isolated tryptic fragments and generally correspond to acidic residues located at either end of each transmembrane helix (e.g., alphaAsp-407). [3H]Azicholesterol labeling was also mapped to peptides that contain either the M3 or M1 segment of each nAChR subunit. These results establish that cholesterol likely interacts with the M4, M3, and M1 segments of each subunit, and therefore, the cholesterol binding domain fully overlaps the lipid-protein interface of the nAChR.     

Topography of helices 5-7 in membrane-inserted diphtheria toxin T domain: identification and insertion boundaries of two hydrophobic sequences that do not form a stable transmembrane hairpin

The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes. T domain hydrophobic helices 8 and 9 can adopt two conformations, one close to the membrane surface (P state) and a second in which they apparently form a transmembrane hairpin (TM state). We have now studied T domain helices 5-7, a second cluster of hydrophobic helices, using Cys-scanning mutagenesis. After fluorescently labeling a series of Cys residues, penetration into a non-polar environment, accessibility to externally added antibodies, and relative depth in the bilayer were monitored. It was found that helices 5-7 insert shallowly in the P state and deeply in the TM state. Thus, the conformational changes in helices 5-7 are both similar and somehow linked to those in helices 8 and 9. The boundaries of deeply inserting sequences were also identified. One deeply inserted segment was found to span residues 270 to 290, which overlaps helix 5, and a second spanned residues 300 to 320, which includes most of helix 6 and all of helix 7. This indicates that helices 6 and 7 form a continuous hydrophobic segment despite their separation by a Pro-containing kink. Additionally, it is found that in the TM state some residues in the hydrophilic loop between helices 5 and 6 become more highly exposed than they are in the P state. Their exposure to external solution in the TM state indicates that helices 5-7 do not form a stable transmembrane hairpin. However, helix 5 and/or helices 6 plus 7 could form transmembrane structures that are in equilibrium with non-transmembrane states, or be kinetically prevented from forming a transmembrane structure. How helices 5-7 might influence the mechanism by which the T domain aids translocation of the diphtheria toxin A chain across membranes is discussed.     

Engineering conformational flexibility in the lactose permease of Escherichia coli: use of glycine-scanning mutagenesis to rescue mutant Glu325-->Asp

Lactose/H(+) symport by lactose permease of Escherichia coli involves interactions between four irreplaceable charged residues in transmembrane helices that play essential roles in H(+) translocation and coupling [Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix IX) with Glu325 (helix X)], as well as Glu126 (helix IV) and Arg144 (helix V) which are obligatory for substrate binding. The conservative mutation Glu325-->Asp causes a 10-fold reduction in the V(max) for active lactose transport and markedly decreased lactose-induced H(+) influx with no effect on exchange or counterflow, neither of which involves H(+) symport. Thus, shortening the side chain may weaken the interaction of the carboxyl group at position 325 with the guanidino group of Arg302. Therefore, Gly-scanning mutagenesis of helices IX and X and the intervening loop was employed systematically with mutant Glu325-->Asp in an effort to rescue function by introducing conformational flexibility between the two helices. Five Gly replacement mutants in the Glu325-->Asp background are identified that exhibit significantly higher transport activity. Furthermore, mutant Val316-->Gly/Glu325-->Asp catalyzes active transport, efflux, and lactose-induced H(+) influx with kinetic properties approaching those of wild-type permease. It is proposed that introduction of conformational flexibility at the interface between helices IX and X improves juxtapositioning between Arg302 and Asp325 during turnover, thereby allowing more effective deprotonation of the permease on the inner surface of the membrane [Sahin-Tóth, M., Karlin, A., and Kaback, H. R. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10729-10732.