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.     

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.     

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 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.     

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.     

Tertiary contacts of helix V in the lactose permease determined by site-directed chemical cross-linking in situ

The six N-terminal transmembrane helices (N6) and the six C-terminal transmembrane helices (C6) in lactose permease, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices V and VII, VIII, or X was studied by thiol-specific chemical cross-linking. The results demonstrate that Cys residues in the periplasmic half of helix V cross-link with Cys residues in the periplasmic half of helix VII. In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices V and VII. Moreover, Cys residues on one entire face of helix V cross-link with Cys residues on one face of helix VIII. Finally, paired Cys residues at the cytoplasmic ends of helices V and X cross-link, but no cross-linking is observed when paired Cys residues are placed at the periplasmic ends of the two helices. Taken together, the results indicate that the periplasmic halves of helices V and VII are in close proximity and that the two helices tilt away from one another toward the cytoplasmic side of the membrane. Furthermore, helices V and VIII are in close proximity throughout their lengths and do not tilt appreciably with respect to one another, and helices V and X are in close proximity at the cytoplasmic but not at the periplasmic face of the membrane.     

Ligand-induced movements of inner transmembrane helices of Glut1 revealed by chemical cross-linking of di-cysteine mutants

The relative orientation and proximity of the pseudo-symmetrical inner transmembrane helical pairs 5/8 and 2/11 of Glut1 were analyzed by chemical cross-linking of di-cysteine mutants. Thirteen functional di-cysteine mutants were created from a C-less Glut1 reporter construct containing cysteine substitutions in helices 5 and 8 or helices 2 and 11. The mutants were expressed in Xenopus oocytes and the sensitivity of each mutant to intramolecular cross-linking by two homobifunctional thiol-specific reagents was ascertained by protease cleavage followed by immunoblot analysis. Five of 9 mutants with cysteine residues predicted to lie in close proximity to each other were susceptible to cross-linking by one or both reagents. None of 4 mutants with cysteine substitutions predicted to lie on opposite faces of their respective helices was susceptible to cross-linking. Additionally, the cross-linking of a di-cysteine pair (A70C/M420C, helices 2/11) predicted to lie near the exoplasmic face of the membrane was stimulated by ethylidene glucose, a non-transported glucose analog that preferentially binds to the exofacial substrate-binding site, suggesting that the binding of this ligand stimulates the closure of helices at the exoplasmic face of the membrane. In contrast, the cross-linking of a second di-cysteine pair (T158C/L325, helices 5/8), predicted to lie near the cytoplasmic face of the membrane, was stimulated by cytochalasin B, a glucose transport inhibitor that competitively inhibits substrate efflux, suggesting that this compound recruits the transporter to a conformational state in which closure of inner helices occurs at the cytoplasmic face of the membrane. This observation provides a structural explanation for the competitive inhibition of substrate efflux by cytochalasin B. These data indicate that the binding of competitive inhibitors of glucose efflux or influx induce occluded states in the transporter in which substrate is excluded from the exofacial or endofacial binding site.     

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helices IV and V that contain the major determinants for substrate binding

Helices IV and V in the lactose permease of Escherichia coli contain the major determinants for substrate binding [Glu126 (helix IV), Arg144 (helix V), and Cys148 (helix V)]. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 48 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in the absence or presence of ligand. In right-side-out membrane vesicles, Cys residues in the cytoplasmic halves of both helices react with NEM in the absence of ligand, while Cys residues in the periplasmic halves do not. Five Cys replacement mutants at the periplasmic end of helix V and one at the cytoplasmic end of helix V label only in the presence of ligand. Interestingly, in addition to native Cys148, a known binding-site residue, labeling of mutant Ala122 --> Cys, which is located in helix IV across from Cys148, is markedly attenuated by ligand. Furthermore, alkylation of the Ala122 --> Cys mutant blocks transport, and protection is afforded by substrate, indicating that Ala122 is also a component of the sugar binding site. Methanethiosulfonate ethylsulfonate, an impermeant thiol reagent shown clearly in this paper to be impermeant in E. coli spheroplasts, was used to identify substituted Cys side chains exposed to water and accessible from the periplasmic side. Most of the Cys mutants in the cytoplasmic halves of helices IV and V, as well as two residues in the intervening loop, are accessible to the aqueous phase from the periplasmic face of the membrane. The findings indicate that the cytoplasmic halves of helices IV and V are more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that exhibit ligand-induced changes are located for the most part in the vicinity of the residues directly involved in substrate binding, as well as the cytoplasmic loop between helices IV and V.     

Structural basis of GLUT1 inhibition by cytoplasmic ATP

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)-mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231-13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1-ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7-8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1-GLUT4 chimera in which loop 6-7 of GLUT1 is substituted with loop 6-7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6-7.     

Amphiphilicity index of polar amino acids as an aid in the characterization of amino acid preference at membrane-water interfaces

MOTIVATION: An amphiphilicity index of amino acid residues was developed for improving the method of transmembrane helix prediction. RESULTS: The transfer energy of a hydrocarbon stem group beyond the gamma-carbon was calculated from the accessible surface area, and used to index the amphiphilicity of the residue. Non-zero amphiphilicity index values were obtained for lysine, arginine, histidine, glutamic acid, glutamine, tyrosine and tryptophan. Those residues were found to be abundant in the end regions of transmembrane helices, indicating their preference for the membrane-water interface. The moving average of the amphiphilicity index actually showed significant peaks in the end regions of most transmembrane helices. A dispersion diagram of average amphiphilicity index versus average hydrophobicity index was devised to facilitate discrimination of transmembrane helices. AVAILABILITY: The amphiphilicity index has been incorporated into a system, SOSUI, for the discrimination of membrane proteins and the prdiction of tranmembrane helical regions (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html).     

Location of helix III in the lactose permease of Escherichia coli as determined by site-directed thiol cross-linking

The six N-terminal transmembrane helices (N(6)) and the six C-terminal transmembrane helices (C(6)) in the lactose permease of Escherichia coli, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices III (position 78, 81, 84, 86, 87, 88, 90, 93, or 96) and VII (position 227, 228, 231, 232, 235, 238, 239, 241, 243, 245, or 246) was examined by using iodine or two rigid homobifunctional thiol-specific cross-linking reagents with different lengths [N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N, N'-p-phenylenedimaleimide (p-PDM; 10 A)]. Cys residues in the periplasmic half of helix III (position 87, 93, or 96) cross-link to Cys residues in the periplasmic half of helix VII (position 235, 238, 239, 241, or 245). In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices III (position 78 or 81) and VII (position 227, 228, 213, 232, or 235). Therefore, the periplasmic halves of helices III and VII are in close proximity, and the helices tilt away from each other toward the cytoplasmic face of the membrane. On the basis of the findings, a modified helix packing model for the permease is presented.     

Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1

Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2-81). Vpu(2-37) encompasses the N-terminal transmembrane alpha-helix; Vpu(2-51) spans the N-terminal transmembrane helix and the first cytoplasmic alpha-helix; Vpu(28-81) includes the entire cytoplasmic domain containing the two C-terminal amphipathic alpha-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane alpha-helix. The C-terminal alpha-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.     

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.     

The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix α6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.     

Site-directed chemical cross-linking demonstrates that helix IV is close to helices VII and XI in the lactose permease

The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease, each containing a single-Cys residue, were coexpressed, and proximity was studied. Paired Cys residues in helices IV (positions 114, 116, 119, 122, 125, or 129) and VII (227, 231, 232, 234, 235, 238, 239, 242, 243, 245, or 246) or XI (350, 353, 354, 357, 361, or 364) were tested for cross-linking in the presence of two rigid homobifunctional thiol-specific cross-linkers, N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N,N'-p-phenylenedimaleimide (p-PDM; 10 A). Cys residues in the middle of helix IV (position 119 or 122) cross-link to Cys residues in the middle of helix VII (position 238, 239, 242, or 243). In contrast, no cross-linking is evident with paired Cys residues at either end of helix IV (position 114, 116, 125, or 129) or helix VII (position 227, 231, 232, 234, 235, 245, or 246). On the other hand, Cys residues in the cytoplasmic half of helix IV (position 125 or 129) cross-link with Cys residues in the cytoplasmic half of helix XI (position 350, 353, or 354), while paired Cys residues at the periplasmic ends of the two helices do not cross-link. The results indicate that helices IV and VII cross in a scissors-like manner with the cytoplasmic end of helix IV tilting toward helix XI.     

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.     

The role of C-terminal ionic residues in self-association of apolipoprotein A-I

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein and is comprised of a helical bundle domain and a C-terminal (CT) domain encompassing the last ~65 amino acid residues of the 243-residue protein. The CT domain contains three putative helices (helix 8, 9, and 10) and is critical for initiating lipid binding and harbors sites that mediate self-association of the lipid-free protein. Three lysine residues reside in helix-8 (K195, 206, 208), and three in helix-10 (K226, 238, 239). To determine the role of each CT lysine residue in apoA-I self-association, single, double and triple lysine to glutamine mutants were engineered via site-directed mutagenesis. Circular dichroism and chemical denaturation analysis revealed all mutants retained their structural integrity. Chemical crosslinking and size-exclusion chromatography showed a small effect on self-association when helix-8 lysine residues were changed into glutamine. In contrast, mutation of the three helix-10 lysine residues resulted in a predominantly monomeric protein and K226 was identified as a critical residue. When helix-10 glutamate residues 223, 234, or 235 were substituted with glutamine, reduced self-association was observed similar to that of the helix-10 lysine variants, suggesting ionic interactions between these residues. Thus, helix-10 is a critical part of apoA-I mediating self-association, and disruption of ionic interactions changes apoA-I from an oligomeric state into a monomer. Since the helix-10 triple mutant solubilized phospholipid vesicles at higher rates compared to wild-type apoA-I, this indicates monomeric apoA-I is more potent in lipid binding, presumably because helix-10 is fully accessible to interact with lipids.     

Cysteine scanning mutagenesis and disulfide mapping studies of the TatA component of the bacterial twin arginine translocase

The Tat (twin arginine translocation) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The integral membrane proteins TatA, TatB, and TatC are essential components of the Tat pathway. TatA forms high order oligomers and is thought to constitute the protein-translocating unit of the Tat system. Cysteine scanning mutagenesis was used to systematically investigate the functional importance of residues in the essential N-terminal transmembrane and amphipathic helices of Escherichia coli TatA. Cysteine substitutions of most residues in the amphipathic helix, including all the residues on the hydrophobic face of the helix, severely compromise Tat function. Glutamine 8 was identified as the only residue in the transmembrane helix that is critical for TatA function. The cysteine variants in the transmembrane helix were used in disulfide mapping experiments to probe the oligomeric arrangement of TatA protomers within the larger TatA complex. Residues in the center of the transmembrane helix (including residues 10-16) show a distinct pattern of cross-linking indicating that this region of the protein forms well defined interactions with other protomers. At least two interacting faces were detected. The results of our TatA studies are compared with analogous data for the homologous, but functionally distinct, TatB protein. This comparison reveals that it is only in TatA that the amphipathic helix is sensitive to amino acid substitutions. The TatA amphipathic helix may play a role in forming and controlling the path of substrate movement across the membrane.     

Application of SCAM (Substituted Cysteine Accessibility Method) to Gap Junction Intercellular Channels

The pore-lining residues of gap junction channels determine their permeability to ions and small cellular metabolites. These residues can be identified through systematic cysteine substitution and accessibility analysis, commonly known as SCAM (Substituted Cysteine Accessibility Method). However, application of this technique to intercellular channels is more complicated than for their transmembrane counterparts. We have utilized a novel dual-oocyte perfusion device to apply cysteine reagents to the cytoplasmic face of paired, voltage-clamped Xenopus oocytes. In this configuration, a large and irreversible cysteine reagent MBB (maliemidobutyryl biocytin, mw 537) was shown to readily traverse the gap junction pore and induce conductance changes upon reaction of accessible sites. Of the 11 reactive sites identified, 6 were located in M3, where they span the bilayer. They display a periodicity characteristic of the tilted helix that lines the pore in the gap junction structure of Unger et al. (1999). Access to several of the other sites was attributed to aqueous crevices between transmembrane helices. Reactive sites were slightly different than those identified for gap junction hemichannels (Zhou et al. 1997), suggesting that conformational changes occur upon docking.