Crystal structures of an O-like blue form and an anion-free yellow form of pharaonis halorhodopsin

Halorhodopsin from Natronomonas pharaonis (pHR) was previously crystallized into a monoclinic space group C2, and the structure of the chloride-bound purple form was determined. Here, we report the crystal structures of two chloride-free forms of pHR, that is, an O-like blue form and an M-like yellow form. When the C2 crystal was soaked in a chloride-free alkaline solution, the protein packing was largely altered and the yellow form containing all-trans retinal was generated. Upon neutralization, this yellow form was converted into the blue form. From structural comparison of the different forms of pHR, it was shown that the removal of a chloride ion from the primary binding site (site I), which is located between the retinal Schiff base and Thr126, is accompanied by such a deformation of helix C that the side chain of Thr126 moves toward helix G, leading to a significant shrinkage of site I. A large structural change is also induced in the chloride uptake pathway, where a flip motion of the side chain of Glu234 is accompanied by large movements of the surrounding aromatic residues. Irrespective of different charge distributions at the active site, there was no large difference in the structures of the yellow form and the blue form. It is shown that the yellow-to-purple transition is initiated by the entrance of one water and one HCl to the active site, where the proton and the chloride ion in HCl are transferred to the Schiff base and site I, respectively.     

Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga

Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light-dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 Å resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the “late proton release”.     

Retinal-Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK_a of 5.8 ± 0.1. It was proposed that this pK_a is associated with the pK_a of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (< 3 M) may be due to an altered conformation adopted by the apomembrane, which is not capable of forming the pigment. Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHR opsin produces pigments with 11-cis retinal and 9-cis retinal owing to the thermal isomerization of these retinal isomers to all-trans retinal. The isomerization rate depends on the pH, and it is faster at a higher pH. The pK_a value of the isomerization process is similar to the pK_a of the binding process of these retinals, which suggests that Lys256 is also involved in the isomerization process. The isomerization is independent of the sodium chloride concentration. However, in the absence of sodium chloride, the apoprotein adopts such a conformation, which does not prevent the isomerization of retinal, but it prevents a covalent bond formation with the lysine residue. The rate and the thermodynamic parameter analysis of the retinal isomerization by NpHR apoprotein led to the conclusion that the apomembrane catalyzes the isomerization via a triplet mechanism.     

Insight to pyrazinamide resistance inMycobacterium tuberculosisby molecular docking

Pyrazinamide (PZA) - an important drug in the anti-tuberculosis therapy, activated by an enzyme Pyrazinamidase (PZase). The basis of PZA resistance in Mycobacterium tuberculosis was owing to mutation in pncA gene coding for PZase. Homology modeling of PZase was performed using software Discovery Studio (DS) 2.0 based on the crystal structure of the PZase from Pyrococcus horikoshii (PDB code 1im5), in this study. The model comprises of one sheet with six parallel strands and seven helices with the amino acids Asp8, Asp49, Trp68, Lys96, Ala134, Thr135 and Cys138 at the active site. Five mutants were generated with Gly at position 8, Thr at position 96, Arg at position 104, Tyr and Ser at position 138. The Wild-type (WT) and five mutant models were docked with PZA. The results indicate that the mutants Lys96Thr, Ser104Arg Asp8Gly and Cys138Tyr may contribute to higher level drug resistance than Cys138Ser. These models provide the first in-silico evidence for the binding interaction of PZA with PZase and form the basis for rationalization of PZA resistance in naturally occurring pncA mutant strains of M. tuberculosis.     

Large Deformation of Helix F during the Photoreaction Cycle of Pharaonis Halorhodopsin in Complex with Azide

Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl⁻ and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (∼4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport.     

Mechanism divergence in microbial rhodopsins

A fundamental design principle of microbial rhodopsins is that they share the same basic light-induced conversion between two conformers. Alternate access of the Schiff base to the outside and to the cytoplasm in the outwardly open “E” conformer and cytoplasmically open “C” conformer, respectively, combined with appropriate timing of pKa changes controlling Schiff base proton release and uptake make the proton path through the pumps vectorial. Phototaxis receptors in prokaryotes, sensory rhodopsins I and II, have evolved new chemical processes not found in their proton pump ancestors, to alter the consequences of the conformational change or modify the change itself. Like proton pumps, sensory rhodopsin II undergoes a photoinduced E → C transition, with the C conformer a transient intermediate in the photocycle. In contrast, one light-sensor (sensory rhodopsin I bound to its transducer HtrI) exists in the dark as the C conformer and undergoes a light-induced C → E transition, with the E conformer a transient photocycle intermediate. Current results indicate that algal phototaxis receptors channelrhodopsins undergo redirected Schiff base proton transfers and a modified E → C transition which, contrary to the proton pumps and other sensory rhodopsins, is not accompanied by the closure of the external half-channel. The article will review our current understanding of how the shared basic structure and chemistry of microbial rhodopsins have been modified during evolution to create diverse molecular functions: light-driven ion transport and photosensory signaling by protein–protein interaction and light-gated ion channel activity.     

The Photocycle and Proton Translocation Pathway in a Cyanobacterial Ion-Pumping Rhodopsin

The genome of thylakoidless cyanobacterium Gloeobacter violaceus encodes a fast-cycling rhodopsin capable of light-driven proton transport. We characterize the dark state, the photocycle, and the proton translocation pathway of GR spectroscopically. The dark state of GR contains predominantly all-trans-retinal and, similar to proteorhodopsin, does not show the light/dark adaptation. We found an unusually strong coupling between the conformation of the retinal and the site of Glu¹³², the homolog of Asp⁹⁶ of BR. Although the photocycle of GR is similar to that of proteorhodopsin in general, it differs in accumulating two intermediates typical for BR, the L-like and the N-like states. The latter state has a deprotonated cytoplasmic proton donor and is spectrally distinct from the strongly red-shifted N intermediate known for proteorhodopsin. The proton uptake precedes the release and occurs during the transition to the O intermediate. The proton translocation pathway of GR is similar to those of other proton-pumping rhodopsins, involving homologs of BR Schiff base proton acceptor and donor Asp⁸⁵ and Asp⁹⁶ (Asp¹²¹ and Glu¹³²). We assigned a pair of FTIR bands (positive at 1749 cm⁻¹ and negative at 1734 cm⁻¹) to the protonation and deprotonation, respectively, of these carboxylic acids.     

Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK_a of the Schiff base (the primary proton donor) and the low pK_a of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

Single replacement constructs of all hydroxyl, basic, and acidic amino acids identify new function and structure-sensitive regions of the mitochondrial phosphate transport protein

The phosphate transport protein (PTP) catalyzes the proton cotransport of phosphate into the mitochondrial matrix. It functions as a homodimer, and thus residues of the phosphate and proton pores are somewhat scattered throughout the primary sequence. With 71 new single mutation per subunit PTPs, all its hydroxyl, basic, and acidic residues have now been replaced to identify these essential residues. We assayed the initial rate of pH gradient-dependent unidirectional phosphate transport activity and the liposome incorporation efficiency (LIE) of these mutants. Single mutations of Thr79, Tyr83, Lys90, Tyr94, and Lys98 inactivate transport. The spacings between these residues imply that they are located along the same face of transmembrane (TM) helix B, requiring an extension of its current model C-terminal domain by 10 residues. This extension superimposes very well onto the shorter bovine PTP helix B, leaving a 15-residue hydrophobic extension of the yeast helix B N-terminus. This is similar to the helix D and F regions of the yeast PTP. Only one transport-inhibiting mutation is located within loops: Ser158Thr in the matrix loop between helices C and D. All other transport-inhibiting mutations are located within the TM helices. Mutations that yield LIEs of <6% are all, except for four, within helices. The four exceptions are Tyr12Ala near the PTP N-terminus and Arg159Ala, Glu163Gln, and Glu164Gln in the loop between helices C and D. The PTP C-terminal segment beyond Thr214 at the N-terminus of helix E has 11 mutations with LIEs >20% and none with LIE <6%. Mutations with LIEs >20% are located near the ends of all the TM helices except TM helix D. Only a few mutations alter PTP structure (LIE) and also affect PTP transport activity. A novel observation is that Ser4Ala blocks the formation of PTP bacterial inclusion bodies.     

Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.     

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.     

Functional significance of a protein conformation change at the cytoplasmic end of helix F during the bacteriorhodopsin photocycle.

The second half of the photocycle of the light-driven proton pump bacteriorhodopsin includes proton transfers between D96 and the retinal Schiff base (the M to N reaction) and between the cytoplasmic surface and D96 (decay of the N intermediate). The inhibitory effects of decreased water activity and increased hydrostatic pressure have suggested that a conformational change resulting in greater hydration of the cytoplasmic region is required for proton transfer from D96 to the Schiff base, and have raised the possibility that the reversal of this process might be required for the subsequent reprotonation of D96 from the cytoplasmic surface. Tilt of the cytoplasmic end of helix F has been suggested by electron diffraction of the M intermediate. Introduction of bulky groups, such as various maleimide labels, to engineered cysteines at the cytoplasmic ends of helices A, B, C, E, and G produce only minor perturbation of the decays of M and N, but major changes in these reactions when the label is linked to helix F. In these samples the reprotonation of the Schiff base is accelerated and the reprotonation of D96 is strongly retarded. Cross-linking with benzophenone introduced at this location, but not at the others, causes the opposite change: the reprotonation of the Schiff base is greatly slowed while the reprotonation of D96 is accelerated. We conclude that, consistent with the structure from diffraction, the proton transfers in the second half of the photocycle are facilitated by motion of the cytoplasmic end of helix F, first away from the center of the protein and then back.     

An internal hydrophobic helical domain of Bacillus subtilis enolase is essential but not sufficient as a non-cleavable signal for its secretion

Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase.     

Crystallographic and Cellular Characterisation of Two Mechanisms Stabilising the Native Fold of α1-Antitrypsin: Implications for Disease and Drug Design

The common Z mutant (Glu342Lys) of α₁-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native α₁-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 Å structure of Thr114Phe α₁-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F-β-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z α₁-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of α₁-antitrypsin and increase secretion of monomeric protein in cell models of disease.     

Structure of bacteriorhodopsin at 1.55 A resolution.

Th?e atomic structure of the light-driven ion pump bacteriorhodopsin and the surrounding lipid matrix was determined by X-ray diffraction of crystals grown in cubic lipid phase. In the extracellular region, an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base and the proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-bulge that causes a non-proline? kink. The bulge is stabilized by hydrogen-bonding of the main-chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The results indicate extensive involvement of bound water molecules in both the structure and the function of this seven-helical membrane protein. A bilayer of 18 tightly bound lipid chains forms an annulus around the protein in the crystal. Contacts between the trimers in the membrane plane are mediated almost exclusively by lipids.     

Inter-helical hydrogen bonds are essential elements for intra-protein signal transduction: the role of Asp115 in bacteriorhodopsin transport function

The behavior of the D115A mutant was analyzed by time-resolved UV-Vis and Fourier transformed infrared (FTIR) spectroscopies, aiming to clarify the role of Asp115 in the intra-protein signal transductions occurring during the bacteriorhodopsin photocycle. UV-Vis data on the D115A mutant show severely desynchronized photocycle kinetics. FTIR data show a poor transmission of the retinal isomerization to the chromoprotein, evidenced by strongly attenuated helical changes (amide I), the remarkable absence of environment alterations and protonation/deprotonation events related to Asp96 and direct Schiff base (SB) protonation form the bulk. This argues for the interactions of Asp115 with Leu87 (via water molecule) and Thr90 as key elements for the effective and vectorial proton path between Asp96 and the SB, in the cytoplasmic half of bacteriorhodopsin. The results strongly suggest the presence of a regulation motif enclosed in helices C and D (Thr90-Pro91/Asp115) which drives properly the dynamics of helix C through a set of interactions. It also supports the idea that intra-helical hydrogen bonding clusters in the buried regions of transmembrane proteins can be potential elements in intra-protein signal transduction.     

Solid-state NMR and functional studies on proteorhodopsin

Proteins of the proteorhodopsin (PR) family are found abundantly in many marine bacteria in the photic zone of the oceans. They are colour-tuned to their environment. The green absorbing species has been shown to act as a light-driven proton pump and thus could form a potential source of energy. The pK_a of the primary proton acceptor is close to the pH of seawater which could also indicate a regulatory role. Here, we review and summarize our own recent findings in the context of known data and present some new results. Proton transfer in vitro by PR is shown by a fluorescence assay which confirms a pH dependent vectoriality. Previously reported low diffracting 2D crystal preparations of PR are assessed for their use for solid-state NMR by two dimensional ¹³C-¹³C DARR spectra. ¹⁵N-¹H HETCOR MAS NMR experiments show bound water in the vicinity of the protonated Schiff base which could play a role in proton transfer. The effect of highly conserved H75 onto the properties of the chromophore has been investigated by single site mutations. They do show a pronounced effect onto the optical absorption maximum and the pK_a of the proton acceptor but have only a small effect onto the ¹⁵N chemical shifts of the protonated Schiff base.     

Proton transport by sensory rhodopsins and its modulation by transducer-binding

The study of light-induced proton transfers in the archaeal sensory rhodopsins (SR), phototaxis receptors in Halobacterium salinarum, has contributed important insights into their mechanism of signaling to their cognate transducer subunits in the signaling complex. Essential features of the bacteriorhodopsin (BR) pumping mechanism have been conserved in the evolution of the sensors, which carry out light-driven electrogenic proton transport when their transducers are removed. The interaction of SRI with its transducer blocks proton-conducting channels in the receptor thereby inhibiting its proton pumping, indicating that the pump machinery, rather than the transport activity itself, is functionally important for signaling. Analysis of SRII mutants has shown that the salt bridge between the protonated Schiff base and its counterion Asp73 constrains the receptor in its inactive conformation. Similarly, in BR, the corresponding salt bridge between the protonated Schiff base and Asp85 contributes to constraining the protein in a conformation in which its cytoplasmic channel is closed. Transducer chimera studies further indicate that the receptor conformational changes are transmitted from the sensors to their cognate transducers through transmembrane helix-helix interaction. These and other results reviewed here support a signaling mechanism in which tilting of helices on the cytoplasmic side (primarily outward tilting of helix F), similar to that which occurs in BR in its open cytoplasmic channel conformation, causes structural alterations in the transducer transmembrane helices.     

High-Resolution Orientation and Depth of Insertion of the Voltage-Sensing S4 Helix of a Potassium Channel in Lipid Bilayers

The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltage-sensing domain. The interaction of these positively charged Arg residues with the lipid membrane has been of intense interest for understanding how membrane proteins fold to allow charged residues to insert into lipid bilayers against free-energy barriers. Using solid-state NMR, we have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers. Two-dimensional ¹⁵N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40° and two possible rotation angles differing by 180° around the helix axis. Remarkably, the tilt angle and one of the two rotation angles are identical to those of the S4 helix in the intact voltage-sensing domain, suggesting that interactions between the S4 segment and other helices of the voltage-sensing domain are not essential for the membrane topology of the S4 helix. ¹³C-³¹P distances between the S4 backbone and the lipid ³¹P indicate a ∼ 9 Å local thinning and 2 Å average thinning of the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphochloline)/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 Å from the guanidinium C^ζ of the second Arg to ³¹P indicates the existence of guanidinium phosphate hydrogen bonding and salt bridges. These data suggest that the structure of the Kv gating helix is mainly determined by protein-lipid interactions instead of interhelical protein-protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix.