Topology and secondary structure of the N-terminal domain of diacylglycerol kinase

Prokaryotic diacylglycerol kinase (DAGK) functions as a homotrimer of 13 kDa subunits, each of which has three transmembrane segments. This enzyme is conditionally essential to some bacteria and serves as a model system for studies of membrane protein biocatalysis, stability, folding, and misfolding. In this work, the detailed topology and secondary structure of DAGK's N-terminus up through the loop following the first transmembrane domain were probed by NMR spectroscopy. Secondary structure was mapped by measuring 13C NMR chemical shifts. Residue-to-residue topology was probed by measuring 19F NMR relaxation rates for site-specifically labeled samples in the presence and absence of polar and hydrophobic paramagnetic probes. Most of DAGK's N-terminal cytoplasmic and first transmembrane segments are alpha-helical. The first and second transmembrane helices are separated by a short loop from residues 48 to 52. The first transmembrane segment extends from residues 32 to 48. Most of the N-terminal cytoplasmic domain lies near the interface but does not extend deeply into the membrane. Finally, catalytic activities measured for the single cysteine mutants before and after chemical labeling suggest that the N-terminal cytoplasmic domain likely contains a number of critical active site residues. The results, therefore, suggest that DAGK's active site lies very near to the water/bilayer interface.     

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.     

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.     

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.     

Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases

The Na(+)-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c(11) rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7A resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c(11) ring of I.tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F(1)F(o) holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na(+). From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5mM NaCl, respectively. These pK values are identical with those determined for the F(1)F(o) complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na(+) from the cytoplasm through access channels within the c-ring itself.     

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.     

Chemical reactivities of cysteine substitutions in subunit a of ATP synthase define residues gating H+ transport from each side of the membrane

Subunit a plays a key role in coupling H(+) transport to rotations of the subunit c-ring in F(1)F(o) ATP synthase. In Escherichia coli, H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F(o) subunit c. Based upon the Ag(+) sensitivity of Cys substituted into subunit a, H(+) are thought to reach Asp-61 via aqueous pathways mapping to surfaces of TMH 2-5. In this study we have extended characterization of the most Ag(+)-sensitive residues in subunit a with cysteine reactive methanethiosulfonate (MTS) reagents and Cd(2+). The effect of these reagents on ATPase-coupled H(+) transport was measured using inside-out membrane vesicles. Cd(2+) inhibited the activity of all Ag(+)-sensitive Cys on the cytoplasmic side of the TMHs, and three of these substitutions were also sensitive to inhibition by MTS reagents. On the other hand, Cd(2+) did not inhibit the activities of substitutions at residues 119 and 120 on the periplasmic side of TMH2, and residues 214 and 215 in TMH4 and 252 in TMH5 at the center of the membrane. When inside-out membrane vesicles from each of these substitutions were sonicated during Cd(2+) treatment to expose the periplasmic surface, the ATPase-coupled H(+) transport activity was strongly inhibited. The periplasmic access to N214C and Q252C, and their positioning in the protein at the a-c interface, is consistent with previous proposals that these residues may be involved in gating H(+) access from the periplasmic half-channel to Asp-61 during the protonation step.     

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.     

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.     

Inhibitory and Anchoring Domains in the ATPase Inhibitor Protein IF1 of Bovine Heart Mitochondrial ATP Synthase

The inhibitor protein IF1 is a basic protein of 84 residues which inhibits the ATPase activity of the mitochondrial FoF1-ATP synthase complex without having any effect on ATP synthesis. Results of cross-linking and limited proteolysis experiments are presented showing that in the intact FoF1 complex "in situ," in the inner membrane of bovine heart mitochondria, the central segment of IF1 (residues 42-58) binds to the alpha and beta subunits of F1 in a pH dependent process, and inhibits the ATPase activity. The C-terminal region of IF1 binds, simultaneously, to the OSCP subunit of Fo in a pH-independent process. This binding keeps IF1 anchored to the complex, both under inhibitory conditions, at acidic pH, and noninhibitory conditions at alkaline pH.     

Sequence and structure patterns in proteins from an analysis of the shortest helices: implications for helix nucleation

The shortest helices (three-length 3(10) and four-length alpha), most abundant among helices of different lengths, have been analyzed from a database of protein structures. A characteristic feature of three-length 3(10)-helices is the shifted backbone conformation for the C-terminal residue (phi,psi angles: -95 degrees,0 degrees ), compared to the rest of the helix (-62 degrees,-24 degrees ). The deviation can be attributed to the release of electrostatic repulsion between the carbonyl oxygen atoms at the two C-terminal residues and further stabilization (due to a more linear geometry) of an intrahelical hydrogen bond. A consequence of this non-canonical C-terminal backbone conformation can be a potential origin of helix kinks when a 3(10)-helix is sequence-contiguous at the alpha-helix N-terminal. An analysis of hydrogen bonding, as well as hydrophobic interactions in the shortest helices shows that capping interactions, some of them not observed for longer helices, dominate at the N termini. Further, consideration of the distribution of amino acid residues indicates that the shortest helices resemble the N-terminal end of alpha-helices rather than the C terminus, implying that the folding of helices may be initiated at the N-terminal end, which does not get propagated in the case of the shortest helices. Finally, pairwise comparison of beta-turns and the shortest helices, based on correlation matrices of site-specific amino acid composition, and the relative abundance of these short secondary structural elements, leads to a helix nucleation scheme that considers the formation of an isolated beta-turn (and not an alpha-turn) as the helix nucleation step, with shortest 3(10)-helices as intermediates between the shortest alpha-helix and the beta-turn. Our results ascribe an important role played by shortest 3(10)-helices in proteins with important structural and folding implications.     

A new solution structure of ATP synthase subunit c from thermophilic Bacillus PS3, suggesting a local conformational change for H+-translocation

In F(o)F(1)-ATP synthase, an oligomer ring of F(o)c subunits acts as a rotary proton channel of the F(o)-proton motor. On the basis of the solution structure of the Escherichia coli F(o)c (EF(o)c) monomer, the rotation of the C-terminal helix coupled with the reorientation of the essential Asp61 side-chain on deprotonation was proposed to drive rotation of the whole c-ring. We have determined the NMR structure of F(o)c from thermophilic Bacillus PS3, TF(o)c, in an organic solvent mixture (chloroform/methanol (3:1, v/v)). Our results showed that, independent of pH, the carboxyl group of the essential Glu56 of TF(o)c protrudes toward the outside of the hairpin, a third orientation that differs from either of the two orientations in EF(o)c. Therefore, it would be inappropriate to draw conclusions about the mechanism of c-ring rotation on the basis of the conformations observed only for EF(o)c. The appearance of different hairpin structures shows that there are multiple energy minima for the hairpin structure in terms of helix rotation and axial displacement. The multiple energy minima may also provide a base for the different oligomeric states in the c-ring structure. A rotation mechanism of the F(o) motor coupled with H(+)-translocation is discussed on the basis of these results and the recently reported crystal structure of the c-ring from Ilyobacter tartaricus Na(+)-ATPase.     

Sequence analysis of cytochrome bd oxidase suggests a revised topology for subunit I

Numerous sequences of the cytochrome bd quinol oxidase (cytochrome bd) have recently become available for analysis. The analysis has revealed a small number of conserved residues, a new topology for subunit I and a phylogenetic tree involving extensive horizontal gene transfer. There are 20 conserved residues in subunit I and two in subunit II. Algorithms utilizing multiple sequence alignments predicted a revised topology for cytochrome bd, adding two transmembrane helices to subunit I to the seven that were previously indicated by the analysis of the sequence of the oxidase from E. coli. This revised topology has the effect of relocating the N-terminus and C-terminus to the periplasmic and cytoplasmic sides of the membrane, respectively. The new topology repositions I-H19, the putative ligand for heme b595, close to the periplasmic edge of the membrane, which suggests that the heme b595/heme d active site of the oxidase is located near the outer (periplasmic) surface of the membrane. The most highly conserved region of the sequence of subunit I contains the sequence GRQPW and is located in a predicted periplasmic loop connecting the eighth and ninth transmembrane helices. The potential importance of this region of the protein was previously unsuspected, and it may participate in the binding of either quinol or heme d. There are two very highly conserved glutamates in subunit I, E99 and E107, within the third transmembrane helix (E. coli cytochrome bd-I numbering). It is speculated that these glutamates may be part of a proton channel leading from the cytoplasmic side of the membrane to the heme d oxygen-reactive site, now placed near the periplasmic surface. The revised topology and newly revealed conserved residues provide a clear basis for further experimental tests of these hypotheses. Phylogenetic analysis of the new sequences of cytochrome bd reveals considerable deviation from the 16sRNA tree, suggesting that a large amount of horizontal gene transfer has occurred in the evolution of cytochrome bd.     

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).     

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.     

Phe(475) and Glu(446) but not Ser(445) participate in ATP-binding to the alpha-subunit of Na(+)/K(+)-ATPase

The ATP-binding site of Na(+)/K(+)-ATPase is localized on the large cytoplasmic loop of the alpha-subunit between transmembrane helices H(4) and H(5). Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser(445), Glu(446), and Phe(475) were proposed to be close to the binding pocket. Replacement of Phe(475) with Trp and Glu(446) with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser(445) with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser(445) is close to the binding site, although it does not participate in binding.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.     

Mechanics of coupling proton movements to c-ring rotation in ATP synthase

F1F0 ATP synthases generate ATP by a rotary catalytic mechanism in which H+ transport is coupled to rotation of an oligomeric ring of c subunits extending through the membrane. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Subunit a of the F0 sector is thought to provide proton access channels to and from aspartyl-61. Here, we summarize new information on the structural organization of Escherichia coli subunit a and the mapping of aqueous-accessible residues in the second, fourth and fifth transmembrane helices (TMHs). Aqueous-accessible regions of these helices extend to both the cytoplasmic and periplasmic surface. We propose that aTMH4 rotates to alternately expose the periplasmic or cytoplasmic half-channels to aspartyl-61 of subunit c during the proton transport cycle. The concerted rotation of interacting helices in subunit a and subunit c is proposed to be the mechanical force driving rotation of the c-rotor, using a mechanism akin to meshed gears.     

Novel inter- and intrasubunit contacts between transport-relevant residues of the homodimeric mitochondrial phosphate transport protein

Ser158 is located near the middle of the matrix loop connecting transmembrane helices C and D of the mitochondrial phosphate transport protein (PTP). The mutant Ser158Thr PTP is transport-inactive. His32 is located near the middle of transmembrane helix A and Thr79 is located 5 residues away from transmembrane helix B and its N-terminal (matrix end). Single site mutant PTPs that have either residue replaced with Ala are transport-inactive. Based on the high resolution structure of a subunit of the bovine ADP/ATP translocase, on sequence similarities between members of the mitochondrial transport protein family, and on the PTP subunit/subunit contact site between transmembrane A helices, it is now suggested that the Ser158 site is at the PTP subunit/subunit contact site. This contact site is essential for keeping the transport cycles catalyzed by the two PTP subunits 180 degrees out of phase. The data also suggest that His32 and Thr79 of the same subunit interact and couple the phosphate and the proton transport paths.     

Moving the pH Gate of the Kir1.1 Inward Rectifier Channel

Both structural and functional studies suggest that pH gating of the inward rectifier potassium (K) channel, Kir1.1 (ROMK), is mediated by the convergence of 4 hydrophobic leucines (one from each subunit) near the cytoplasmic bundle-crossing of the inner transmembrane helices. We tested this hypothesis by moving the putative leucine gate from the L160-Kir1.1b to other positions along the inner transmembrane helix, and measuring inward current and conductance as functions of internal pH, using the Xenopus oocyte heterologous expression system. Results of these studies indicated that it was possible to replace the putative inward rectifier pH gate at L160-Kir1.1b by either a leucine or methionine at 157-Kir1.1b (G157L-L160G or G157M-L160G). Although both leucine and methionine gated the channel at 157-Kir1.1b, residues of similar hydrophobicity (tyrosine and valine) did not. Hence, hydrophobicity was a necessary but not a sufficient condition for steric gating at 157. This was in contrast to the 160-Kir1.1b locus, where side-chain hydrophobicity was both a necessary and sufficient property for steric gating. Homology models were constructed for all mutants that expressed significant whole-cell currents, using the closed-state coordinates of the prokaryotic inward rectifier, KirBac1.1. Models of mutants that retained pH gating were too narrow at the bundle crossing to permit hydrated K ion permeation in the closed-state. On the other hand, mutants that lost pH gating had ample space at the bundle crossing for hydrated K permeation in the closed-state. These results support our hypothesis that hydrophobic leucines at the cytoplasmic end of the inner transmembrane helices comprise the principal pH gate of Kir1.1, a gate that can be relocated from 160-Kir1.1b to 157-Kir1.1b.