Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix VII

Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.     

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.     

Site-directed alkylation of cysteine replacements in the lactose permease of Escherichia coli: helices I, III, VI, and XI

To complete a study on site-directed alkylation of Cys replacements in the lactose permease of Escherichia coli (LacY), the reactivity of single-Cys mutants in helices I, III, VI, and XI, as well as some of the adjoining loops, with N-[14C]ethylmaleimide (NEM) or methanethiosulfonate ethylsulfonate (MTSES) was studied in right-side-out membrane vesicles. With the exception of several positions in the middle of helix I, which either face the bilayer or are in close proximity to other helices, the remaining Cys replacements react with the membrane-permeant alkylating agent NEM. In helices III and XI, most Cys replacements are also alkylated by NEM except for positions that face the bilayer. The reactivity of Cys replacements in helix VI is noticeably lower and only 45% of the replacements label. Binding of sugar leads to significant increases in the reactivity of Cys residues that are located primarily at the same level as the sugar-binding site or in the periplasmic half of each helix. Remarkably, studies with small, impermeant MTSES show that single-Cys replacements in the cytoplasmic portions of helices I and XI, which line the inward-facing cavity, are accessible to solvent from the periplasmic surface of the membrane. Moreover, addition of ligand results in increased accessibility of Cys residues to the aqueous milieu in the periplasmic region of the helices, which may reflect structural rearrangements leading to opening of an outward-facing cavity. The findings are consistent with the X-ray structure of LacY and with the alternating access model [Abramson, J., Smirnova, I., et al. (2003) Science 301, 610-615].     

Thiol cross-linking of transmembrane domains IV and V in the lactose permease of Escherichia coli

Glu126 (helix IV) and Arg144 (helix V) in the lactose permease of Escherichia coli are critical for substrate binding and transport, and the two residues are in close proximity and charge-paired. By using a functional permease construct with two tandem factor Xa protease sites in the cytoplasmic loop between helices IV and V, it is shown here that Cys residues in place of Glu126 and Arg144, as well as Ala122 and Val149, spontaneously form disulfide bonds in situ, indicating that this region of transmembrane domains IV and V is in the alpha-helical conformation. To determine if the local structure or environment is perturbed by the presence of an unpaired charge, either Glu126 or Arg144 or both were replaced with Ala, and cross-linking between the Cys pair Ala122-->Cys/Val149-->Cys was studied. Ala replacement for Arg144 causes a marked decrease in cross-linking, while Ala replacement for Glu126 alone or for both Glu126 and Arg144 has little effect. The data provide strong support for the argument that Glu126 and Arg144 are within close proximity and suggest that an unpaired carboxylate at position 126 causes a structural change at the interface between helices IV and V.     

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix X

Helix X in the lactose permease of Escherichia coli contains two residues that are irreplaceable with respect to active transport, His322 and Glu325, as well as Lys319, which is charge-paired with Asp240 in helix VII. Structural and dynamic features of transmembrane helix X are investigated here by site-directed thiol modification of 14 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in right-side-out membrane vesicles. Permease mutants with a Cys residue at position 326, 327, 329, 330, or 331 in the cytoplasmic half of the transmembrane domain are alkylated by NEM at 25 degrees C, a mutant with Cys at position 315 at the periplasmic surface is labeled in the presence of substrate exclusively, and mutants with Cys at positions 317, 318, 320, 321, 324, 328, 332, or 333 do not react with NEM under the conditions tested. Binding of substrate causes increased labeling of a Cys residue at position 315 and decreased labeling of Cys residues at positions 326, 327, and 329. Studies with methanethiosulfonate ethylsulfonate indicate that Cys residues at positions 326, 329, 330, and 331 in the cytoplasmic half are accessible to the aqueous phase from the periplasmic face of the membrane. Ligand binding results in clear attenuation of solvent accessibility of Cys at position 326 and a marginal increase in accessibility of Cys at position 327 to solvent. The findings indicate that the cytoplasmic half of helix X is more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that reflect ligand-induced conformational changes are located on the same face of helix X as Lys319, His322, and Glu325.     

The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.     

Aromatic stacking in the sugar binding site of the lactose permease

Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.     

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.     

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.     

Helices VII and X in the lactose permease of Escherichia coli: proximity and ligand-induced distance changes.

By using functional lactose permease devoid of native Cys residues with a discontinuity in the periplasmic loop between helices VII and VIII (N(7)/C(5) split permease), cross-linking between engineered paired Cys residues in helices VII and X was studied with the homobifunctional, thiol-specific cross-linkers 1,1-methanediyl bismethanethiosulfonate (3 A), N,N'-o- phenylenedimaleimide (6 A) and N,N'-p-phenylenedimaleimide (10 A). Mutant Asp240-->Cys (helix VII)/Lys319-->Cys (helix X) cross-links most efficiently with the 3 A reagent, providing direct support for studies indicating that Asp240 and Lys319 are in close proximity and charge paired. Furthermore, cross-linking the two positions inactivates the protein. Other Cys residues more disposed towards the middle of helix VII cross-link to Cys residues in the approximate middle of helix X, while no cross-linking is evident with paired Cys residues at the periplasmic or cytoplasmic ends of these helices. Thus, helices VII and X are in close proximity in the middle of the membrane. In the presence of ligand, the distance between Cys residues at positions 240 (helice VII) and 319 (helix X) increases. In contrast, the distance between paired Cys residues more disposed towards the cytoplasmic face of the membrane decreases in a manner suggesting that ligand binding induces a scissors-like movement between the two helices. The results are consistent with a recently proposed mechanism for lactose/H(+) symport in which substrate binding induces a conformational change between helices VII and X, during transfer of H(+) from His322 (helix X)/Glu269 (helix VIII) to Glu325 (helix X).     

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.     

Proximity relationships between helices I and XI or XII in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking.

The lactose permease of Escherichia coli was expressed in two fragments (split permease), each with a Cys residue, and cross-linking was studied. Split permease with a discontinuity in either loop II/III (N2C10permease) or loop VI/VII (N6C6permease) was used. Proximity of multiple pairs of Cys residues in helices I and XI or XII was examined by using three homobifunctional thiol-specific cross-linking reagents of different lengths and flexibilities (6 A, rigid; 10 A, rigid; 16 A, flexible) or iodine. Cys residues in the periplasmic half of helix I cross-link to Cys residues in the periplasmic half of helix XI. In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices I and XI. Therefore, the periplasmic halves of helices I and XI are in close proximity, and the helices tilt away from each other towards the cytoplasmic face of the membrane. Cross-linking is also found with paired Cys residues near the middle of helices I and XII, but not with paired Cys residues near either end of the helices. Thus, helices I and XII are in close proximity only in the approximate middle of the membrane. Based on the findings, a modified helix packing model is proposed.     

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.     

Residues Gating the Periplasmic Pathway of LacY

X-ray crystal structures of LacY (lactose permease of Escherichia coli) exhibit a large cytoplasmic cavity containing the residues involved in sugar binding and H⁺ translocation at the apex and a tightly packed side facing the periplasm. However, biochemical and biophysical evidence provide a strong indication that a hydrophilic pathway opens on the external surface of LacY with closing of the cytoplasmic side upon sugar binding. Thus, an alternating-access mechanism in which sugar- and H⁺-binding sites at the approximate middle of the molecule are alternatively exposed to either side of the membrane is likely to underlie LacY-catalyzed sugar/H⁺ symport. To further investigate periplasmic opening, we replaced paired residues on the tightly packed periplasmic side of LacY with Cys, and the effect of cross-linking was studied by testing the accessibility/reactivity of Cys148 with the elongated (∼ 29 Å), impermeant hydrophilic reagent maleimide-PEG2-biotin. When the paired-Cys mutant Ile40 → Cys/Asn245 → Cys containing native Cys148 is oxidized to form a disulfide bond, the reactivity of Cys148 is markedly inhibited. Moreover, the reactivity of Cys148 in this mutant increases with the length of the cross-linking agent. In contrast, maleimide-PEG2-biotin reactivity of Cys148 is unaffected by oxidation of two other paired-Cys mutants at the mouth of the periplasmic cavity. The data indicate that residues Ile40 and Asn245 play a primary role in gating the periplasmic cavity and provide further support for the alternating-access model.     

Site-directed alkylation studies with LacY provide evidence for the alternating access model of transport

In total, 59 single Cys-replacement mutants in helix VII and helix X of the lactose permease of Escherichia coli were subjected to site-directed fluorescence labeling in right-side-out membrane vesicles to complete the testing of Cys accessibility or reactivity. For both helices, accessibility/reactivity is relatively low at the level of the sugar-binding site where the helices are tightly packed. However, labeling of Cys substitutions in helix VII with tetramethylrhodamine-5-maleimide decreases from the middle toward the cytoplasmic end and increases toward the periplasmic end. Helix X is labeled mainly on the side facing the central hydrophilic cavity with relatively small or no changes in the presence of ligand. In contrast, sugar binding causes a significant increase in accessibility/reactivity at the periplasmic end of helix VII. When considered with similar findings from N-ethylmaleimide alkylation studies, the results confirm and extend support for the alternating access model.     

Glutamates 99 and 107 in transmembrane helix III of subunit I of cytochrome bd are critical for binding of the heme b595-d binuclear center and enzyme activity

Cytochrome bd is a quinol oxidase of Escherichia coli under microaerophilic growth conditions. Coupling of the release of protons to the periplasm by quinol oxidation to the uptake of protons from the cytoplasm for dioxygen reduction generates a proton motive force. On the basis of sequence analysis, glutamates 99 and 107 conserved in transmembrane helix III of subunit I have been proposed to convey protons from the cytoplasm to heme d at the periplasmic side. To probe a putative proton channel present in subunit I of E. coli cytochrome bd, we substituted a total of 10 hydrophilic residues and two glycines conserved in helices I and III-V and examined effects of amino acid substitutions on the oxidase activity and bound hemes. We found that Ala or Leu mutants of Arg9 and Thr15 in helix I, Gly93 and Gly100 in helix III, and Ser190 and Thr194 in helix V exhibited the wild-type phenotypes, while Ala and Gln mutants of His126 in helix IV retained all hemes but partially lost the activity. In contrast, substitutions of Thr26 in helix I, Glu99 and Glu107 in helix III, Ser140 in helix IV, and Thr187 in helix V resulted in the concomitant loss of bound heme b558 (T187L) or b595-d (T26L, E99L/A/D, E107L/A/D, and S140A) and the activity. Glu99 and Glu107 mutants except E107L completely lost the heme b595-d center, as reported for heme b595 ligand (His19) mutants. On the basis of this study and previous studies, we propose arrangement of transmembrane helices in subunit I, which may explain possible roles of conserved hydrophilic residues within the membrane.     

Helix Dynamics in LacY: Helices II and IV

Biochemical and biophysical studies based upon crystal structures of both a mutant and wild-type lactose permease from Escherichia coli (LacY) in an inward-facing conformation have led to a model for the symport mechanism in which both sugar and H⁺ binding sites are alternatively accessible to both sides of the membrane. Previous findings indicate that the face of helix II with Asp68 is important for the conformational changes that occur during turnover. As shown here, replacement of Asp68 at the cytoplasmic end of helix II, particularly with Glu, abolishes active transport but the mutants retain the ability to bind galactopyranoside. In the x-ray structure, Asp68 and Lys131 (helix IV) lie within ∼ 4.2 Å of each other. Although a double mutant with Cys replacements at both position 68 and position 131 cross-links efficiently, single replacements for Lys131 exhibit very significant transport activity. Site-directed alkylation studies show that sugar binding by the Asp68 mutants causes closure of the cytoplasmic cavity, similar to wild-type LacY; however, strikingly, the probability of opening the periplasmic pathway upon sugar binding is markedly reduced. Taken together with results from previous mutagenesis and cross-linking studies, these findings lead to a model in which replacement of Asp68 blocks a conformational transition involving helices II and IV that is important for opening the periplasmic cavity. Evidence suggesting that movements of helices II and IV are coupled functionally with movements in the pseudo-symmetrically paired helices VIII and X is also presented.     

Helix packing in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking: helix I is close to helices V and XI

Coexpression of lacY gene fragments encoding the first two transmembrane domains and the remaining 10 transmembrane domains complement in the membrane and catalyze active lactose transport [Wrubel, W., Stochaj, U., et al. (1990) J. Bacteriol. 172, 5374-5381]. Accordingly, a plasmid encoding contiguous, nonoverlapping permease fragments with a discontinuity in the cytoplasmic loop between helices II and III (loop II/III) was constructed (N2C10 permease). When Phe27 (helix I) is replaced with Cys, cross-linking is observed with two native Cys residues, Cys148 (helix V) and Cys355 (helix XI). Cross-linking of a Cys residue at position 27 to Cys148 occurs with N,N'-o-phenylenedimaleimide (o-PDM; rigid 6 A), with N,N'-p-phenylenedimaleimide (p-PDM; rigid 10 A), or with 1,6-bis(maleimido)hexane (BMH; flexible 16 A). On the other hand, with the Phe27-->Cys/Cys355 pair, cross-linking is observed with p-PDM or BMH but not o-PDM. In neither case is cross-linking observed with iodine. It is suggested that a Cys residue at position 27 is within 6-10 A from Cys148 and about 10 A from Cys355. The results provide evidence for proximity between helix I and helices V or XI in the tertiary structure of the permease. In addition, the findings are consistent with other results [Venkatesan, P., Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] indicating that Glu126 (helix IV) and Arg144 (helix V) are within the membrane, rather than at the membrane-water interface on the cytoplasmic face.     

Substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae

We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-β,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.     

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.