Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.     

A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase

The H,K-adenosine triphosphatase (ATPase) of gastric parietal cells is targeted to a regulated membrane compartment that fuses with the apical plasma membrane in response to secretagogue stimulation. Previous work has demonstrated that the alpha subunit of the H, K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the beta subunit carries the signal responsible for the cessation of acid secretion through the retrieval of the pump from the surface to the regulated intracellular compartment. By analyzing the sorting behaviors of a number of chimeric pumps composed of complementary portions of the H, K-ATPase alpha subunit and the highly homologous Na,K-ATPase alpha subunit, we have identified a portion of the gastric H,K-ATPase, which is sufficient to redirect the normally basolateral Na,K-ATPase to the apical surface in transfected epithelial cells. This motif resides within the fourth of the H,K-ATPase alpha subunit's ten predicted transmembrane domains. Although interactions with glycosphingolipid-rich membrane domains have been proposed to play an important role in the targeting of several apical membrane proteins, the apically located chimeras are not found in detergent-insoluble complexes, which are typically enriched in glycosphingolipids. Furthermore, a chimera incorporating the Na, K-ATPase alpha subunit fourth transmembrane domain is apically targeted when both of its flanking sequences derive from H,K-ATPase sequence. These results provide the identification of a defined apical localization signal in a polytopic membrane transport protein, and suggest that this signal functions through conformational interactions between the fourth transmembrane spanning segment and its surrounding sequence domains.     

Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel.

The rapid translocation of H+ along a chain of hydrogen-bonded water molecules, or proton wire, is thought to be an important mechanism for proton permeation through transmembrane channels. Computer simulations are used to study the properties of the proton wire formed by the single-file waters in the gramicidin A channel. The model includes the polypeptidic dimer, with 22 water molecules and one excess proton. The dissociation of the water molecules is taken into account by the "polarization model" of Stillinger and co-workers. The importance of quantum effects due to the light mass of the hydrogen nuclei is examined with the use of discretized Feynman path integral molecular dynamics simulations. Results show that the presence of an excess proton in the pore orients the single-file water molecules and affects the geometry of water-water hydrogen bonding interactions. Rather than a well-defined hydronium ion OH3+ in the single-file region, the protonated species is characterized by a strong hydrogen bond resembling that of O2H5+. The quantum dispersion of protons has a small but significant effect on the equilibrium structure of the hydrogen-bonded water chain. During classical trajectories, proton transfer between consecutive water molecules is a very fast spontaneous process that takes place in the subpicosecond time scale. The translocation along extended regions of the chain takes place neither via a totally concerted mechanism in which the donor-acceptor pattern would flip over the entire chain in a single step, nor via a succession of incoherent hops between well-defined intermediates. Rather, proton transfer in the wire is a semicollective process that results from the subtle interplay of rapid hydrogen-bond length fluctuations along the water chain. These rapid structural fluctuations of the protonated single file of waters around an average position and the slow movements of the average position of the excess proton along the channel axis occur on two very different time scales. Ultimately, it is the slow reorganization of hydrogen bonds between single-file water molecules and channel backbone carbonyl groups that, by affecting the connectivity and the dynamics of the single-file water chain, also limits the translocation of the proton across the pore.     

Effect of diamide on proton translocation by the mitochondrial H+-ATPase

Treatment of sonic submitochondrial particles with the bifunctional thiol reagent, diamide, results in an enhancement of proton conductivity and ATPase activity, which is reversed by the reducing agent dithiothreitol, is suppressed by Fo inhibitors like oligomycin and is absent in particles that are deprived of peripheral Fo polypeptides. The effect of diamide is apparently due to oxidation of dithiols to disulfides in peripheral polypeptide(s) of Fo.     

Structure of the integrin alphaIIb transmembrane segment

Integrin cell-adhesion receptors transduce signals bidirectionally across the plasma membrane via the single-pass transmembrane segments of each alpha and beta subunit. While the beta3 transmembrane segment consists of a linear 29-residue alpha-helix, the structure of the alphaIIb transmembrane segment reveals a linear 24-residue alpha-helix (Ile-966 -Lys-989) followed by a backbone reversal that packs Phe-992-Phe-993 against the transmembrane helix. The length of the alphaIIb transmembrane helix implies the absence of a significant transmembrane helix tilt in contrast to its partnering beta3 subunit. Sequence alignment shows Gly-991-Phe-993 to be fully conserved among all 18 human integrin alpha subunits, suggesting that their unusual structural motif is prototypical for integrin alpha subunits. The alphaIIb transmembrane structure demonstrates a level of complexity within the membrane that is beyond simple transmembrane helices and forms the structural basis for assessing the extent of structural and topological rearrangements upon alphaIIb-beta3 association, i.e. integrin transmembrane signaling.     

Molecular characterization of the yeast vacuolar H+-ATPase proton pore

The Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is composed of at least 13 polypeptides organized into two distinct domains, V(1) and V(0), that are structurally and mechanistically similar to the F(1)-F(0) domains of the F-type ATP synthases. The peripheral V(1) domain is responsible for ATP hydrolysis and is coupled to the mechanism of proton translocation. The integral V(0) domain is responsible for the translocation of protons across the membrane and is composed of five different polypeptides. Unlike the F(0) domain of the F-type ATP synthase, which contains 12 copies of a single 8-kDa proteolipid, the V-ATPase V(0) domain contains three proteolipid species, Vma3p, Vma11p, and Vma16p, with each proteolipid contributing to the mechanism of proton translocation (Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1997) J. Biol. Chem. 272, 4795-4803). Experiments with hemagglutinin- and c-Myc epitope-tagged copies of the proteolipids revealed that each V(0) complex contains all three species of proteolipid with only one copy each of Vma11p and Vma16p but multiple copies of Vma3p. Since the proteolipids of the V(0) complex are predicted to possess four membrane-spanning alpha-helices, twice as many as a single F-ATPase proteolipid subunit, only six V-ATPase proteolipids would be required to form a hexameric ring-like structure similar to the F(0) domain. Therefore, each V(0) complex will likely be composed of four copies of the Vma3p proteolipid in addition to Vma11p and Vma16p. Structural differences within the membrane-spanning domains of both V(0) and F(0) may account for the unique properties of the ATP-hydrolyzing V-ATPase compared with the ATP-generating F-type ATP synthase.     

Conformation of a peptide encompassing the proton translocation channel of vacuolar H(+)-ATPase

The structural properties of a crucial transmembrane helix for proton translocation in vacuolar ATPase are studied using double site-directed spin-labeling combined with electron spin resonance (ESR) (or electron paramagnetic resonance) and circular dichroism spectroscopy in sodium dodecyl sulfate micelles. For this purpose, we use a synthetic peptide derived from transmembrane helix 7 of subunit a from the yeast Saccharomyces cerevisiae vacuolar proton-translocating ATPase that contains two natural cysteine residues suitable for spin-labeling. The interspin distance is calculated using a second-moment analysis of the methanethiosulfonate spin-label ESR spectra at 150 K. Molecular dynamics simulation is used to study the effect of the side-chain dynamics and backbone dynamics on the interspin distance. Based on the combined results from ESR, circular dichroism, and molecular dynamics simulation we conclude that the peptide forms a dynamic alpha-helix. We discuss this finding in the light of current models for proton translocation. A novel role for a buried charged residue (H729) is proposed.     

Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H(+)-ATPase

Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P(2)-type ATPases, M8 has until now received the least attention. The present study has used alanine-scanning mutagenesis to map structure-function relationships throughout M8 of the yeast plasma-membrane H(+)-ATPase. Mutant forms of the ATPase were expressed in secretory vesicles and at the plasma membrane for measurements of ATP hydrolysis and ATP-dependent H(+) pumping. In secretory vesicles, Ala substitutions at a cluster of four positions near the extracytoplasmic end of M8 led to partial uncoupling of H(+) transport from ATP hydrolysis, while substitution of Ser-800 (close to the middle of M8) by Ala increased the apparent stoichiometry of H(+) transport. A similar increase has previously been reported following the substitution of Glu-803 by Gln (Petrov, V. et al., J. Biol. Chem. 275:15709-15718, 2000) at a position known to contribute directly to Ca(2+) binding in the Ca(2+)-ATPase of sarcoplasmic reticulum (Toyoshima, C., et al., Nature 405: 647-655, 2000). Four other mutations in M8 interfered with H(+)-ATPase folding and trafficking to the plasma membrane; based on homology modeling, they occupy positions that appear important for the proper bundling of M8 with M5, M6, M7, and M10. Taken together, these results point to a key role for M8 in the biogenesis, stability, and physiological functioning of the H(+)-ATPase.     

Segment TM7 from the cytoplasmic hemi-channel from VO-H+-V-ATPase includes a flexible region that has a potential role in proton translocation

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY (1)H-(1)H NMR spectra are obtained of sMTM7 dissolved in d(6)-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two alpha-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.     

Monomers of the Neurospora plasma membrane H+-ATPase catalyze efficient proton translocation

Liposomes prepared by sonication of asolectin were fractionated by glycerol density gradient centrifugation, and the small liposomes contained in the upper region of the gradients were used for reconstitution of purified, radiolabeled Neurospora plasma membrane H+-ATPase molecules by our previously published procedures. The reconstituted liposomes were then subjected to two additional rounds of glycerol density gradient centrifugation, which separate the H+-ATPase-bearing proteoliposomes from ATPase-free liposomes by virtue of their greater density. The isolated H+-ATPase-bearing proteoliposomes in two such preparations exhibited a specific H+-ATPase activity of about 11 mumol of Pi liberated/mg of protein/min, which was approximately doubled in the presence of nigericin plus K+, indicating that a large percentage of the H+-ATPase molecules in both preparations were capable of generating a transmembrane protonic potential difference sufficient to impede further proton translocation. Importantly, quantitation of the number of 105,000-dalton ATPase monomers and liposomes in the same preparations by radioactivity determination and counting of negatively stained images in the electron microscope indicated ATPase monomer to liposome ratios of 0.97 and 1.06. Because every liposome in the preparations must have had at least one ATPase monomer, these ratios indicate that very few of the liposomes had more than one, and simple calculations show that the great majority of active ATPase molecules in the preparations must have been present as proton-translocating monomers. The results thus clearly demonstrate that 105,000-dalton monomers of the Neurospora plasma membrane H+-ATPase can catalyze efficient ATP hydrolysis-driven proton translocation.     

The transmembrane domain and the proton channel in proton-pumping transhydrogenases

Proton-pumping nicotinamide nucleotide transhydrogenases are composed of three main domains, the NAD(H)-binding and NADP(H)-binding hydrophilic domains I (dI) and III (dIII), respectively, and the hydrophobic domain II (dII) containing the assumed proton channel. dII in the Escherichia coli enzyme has recently been characterised with regard to topology and a packing model of the helix bundle in dII is proposed. Extensive mutagenesis of conserved charged residues of this domain showed that important residues are betaHis91 and betaAsn222. The pH dependence of betaH91D, as well as betaH91C (unpublished), when compared to that of wild type shows that reduction of 3-acetylpyridine-NAD(+) by NADPH, i.e., the reverse reaction, is optimal at a pH essentially coinciding with the pK(a) of the residue in the beta91 position. It is therefore concluded that the wild-type transhydrogenase is regulated by the degree of protonation of betaHis91. The mechanisms of the interactions between dI+dIII and dII are suggested to involve pronounced conformational changes in a 'hinge' region around betaR265.     

Alanine-scanning mutagenesis of the sixth transmembrane segment of gastric H+,K+-ATPase alpha-subunit

The sixth transmembrane (M6) segment of the catalytic subunit plays an important role in the ion recognition and transport in the type II P-type ATPase families. In this study, we singly mutated all amino acid residues in the M6 segment of gastric H(+),K(+)-ATPase alpha-subunit with alanine, expressed the mutants in HEK-293 cells, and studied the effects of the mutation on the functions of H(+),K(+)-ATPase; overall K(+)-stimulated ATPase, phosphorylation, and dephosphorylation. Four mutants, L819A, D826A, I827A, and L833A, completely lost the K(+)-ATPase activity. Mutant L819A was phosphorylated but hardly dephosphorylated in the presence of K(+), whereas mutants D826A, I827A, and L833A were not phosphorylated from ATP. We found that almost all of these amino acid residues, which are important for the function, are located on the same side of the alpha-helix of the M6 segment. In addition, we found that amino acids involved in the phosphorylation are located exclusively in the cytoplasmic half of the M6 segment and those involved in the K(+)-dependent dephosphorylation are in the luminal half. Several mutants such as I821A, L823A, T825A, and P829A partly retained the K(+)-ATPase activity accompanying the decrease in the rate of phosphorylation.     

Conserved Asp684 in transmembrane segment M6 of the plant plasma membrane P-type proton pump AHA2 is a molecular determinant of proton translocation

The mechanism of proton pumping by P-type H(+)-ATPases is still unclear. In the plant P-type plasma membrane H(+)-ATPase AHA2, two charged residues, Arg(655) and Asp(684), are conserved in transmembrane segments M5 and M6, respectively, a region that has been shown be contribute to ion coordination in related P-type ATPases. Substitution of Arg(655) with either alanine or aspartate resulted in mutant enzymes exhibiting a significant shift in the P-type ATPase E(1)P-E(2)P conformational equilibrium. The mutant proteins accumulated in the E(1)P conformation, but were capable of conducting proton transport. This points to an important role of Arg(655) in the E(1)P-E(2)P conformational transition. The presence of a carboxylate moiety at position Asp(684) proved essential for coupling between initial proton binding and proton pumping. The finding that the carboxylate side chain of Asp(684) contributes to the proton-binding site and appears to function as an absolutely essential proton acceptor along the proton transport pathway is discussed in the context of a possible proton pumping mechanism of P-type H(+)-ATPases.     

Real-time transmembrane translocation of penetratin driven by light-generated proton pumping

Cell penetrating peptides (CPPs) are small peptides that are able to penetrate the plasma membrane of mammalian cells. Because these peptides can also carry large hydrophilic cargos such as proteins, they could potentially be used to transport biologically active drugs across cell membranes to modulate in vivo biology. One characteristic feature of the CPPs is that they typically have a net positive charge. Therefore, a key issue associated with the transport mechanism is the role of the transmembrane electrochemical potential in driving the peptides across the membrane. In this study, we have reconstituted bacteriorhodopsin (bR) in large unilamellar vesicles (LUVs) with fluorescein-labeled CPP penetratin enclosed within the LUVs under conditions when the fluorescence is quenched. Illumination of the bacteriorhodopsin-containing LUVs resulted in creation of a transmembrane proton electrochemical gradient (positive on the inside). Upon generation of this gradient, an increase in fluorescence was observed, which shows that the proton gradient drives the translocation of penetratin. The mechanism most likely can be generalized to other CPPs.     

Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H+K+-ATPase

Summary Membrane fragments containing the H⁺K^–-ATPase from parietal cells have been adsorbed to a planar lipid membrane. The transport activity of the enzyme was determined by measuring electrical currents via the capacitive coupling between the membrane sheets and the planar lipid film. To initiate the pump currents by the ATPase a light-driven concentration jump of ATP from caged ATP was applied as demonstrated previously for Na⁺K⁺-ATPase (Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985.EMBO J. 4:3079–3085). Since H⁺K⁺-ATPase is an electroneutrally working enzyme no stationary pump currents were observed in the presence of K⁺. By separation of the H⁺ and K⁺ transport steps of the reaction cycle, however, the electrogenic step of the phosphorylation could be measured. This was achieved in the absence of K⁺ or at low concentrations of K⁺. The observed transient current is ATP dependent which can be assigned to the proton movement during the phosphorylation. From this it was conclueded that the K⁺ transport during dephosphorylation is electrogenic, too, in contrast to the Na⁺K⁺-ATPase where the K⁺ step is electroneutral. The transient current was measured at different ionic conditions and could be blocked by vanadate and by the H⁺K⁺-ATPase specific inhibitor omeprazole. An alternative mechanism for activation of this inhibitor is discussed.     

Structural and functional characterization of transmembrane segment VII of the Na+/H+ exchanger isoform 1

The Na(+)/H(+) exchanger isoform 1 is an integral membrane protein that regulates intracellular pH by exchanging one intracellular H(+) for one extracellular Na(+). It is composed of an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the structural and functional aspects of the critical transmembrane segment VII (TM VII, residues 251-273) by using alanine scanning mutagenesis and high resolution NMR. Each residue of TM VII was mutated to alanine, the full-length protein expressed, and its activity characterized. TM VII was sensitive to mutation. Mutations at 13 of 22 residues resulted in severely reduced activity, whereas other mutants exhibited varying degrees of decreases in activity. The impaired activities sometimes resulted from low expression and/or low surface targeting. Three of the alanine scanning mutant proteins displayed increased, and two displayed decreased resistance to the Na(+)/H(+) exchanger isoform 1 inhibitor EMD87580. The structure of a peptide of TM VII was determined by using high resolution NMR in dodecylphosphocholine micelles. TM VII is predominantly alpha-helical, with a break in the helix at the functionally critical residues Gly(261)-Glu(262). The relative positions and orientations of the N- and C-terminal helical segments are seen to vary about this extended segment in the ensemble of NMR structures. Our results show that TM VII is a critical transmembrane segment structured as an interrupted helix, with several residues that are essential to both protein function and sensitivity to inhibition.     

Sequence determinants of a transmembrane proton channel: an inverse relationship between stability and function

The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.     

Structure and pH sensitivity of the transmembrane segment 3 of rhodopsin

Activation of G protein-coupled receptors (GPCRs) originates in ligand-induced protein conformational changes that are transmitted to the cytosolic receptor surface. In the photoreceptor rhodopsin, and possibly other rhodopsin-like GPCRs, protonation of a carboxylic acid in the conserved E(D)RY motif at the cytosolic end of transmembrane helix 3 (TM3) is coupled to receptor activation. Here, we have investigated the structure of synthetic peptides derived from rhodopsin TM3. Polarized FTIR spectroscopy reveals a helical structure of a 31-mer TM3 peptide reconstituted into PC vesicles with a large tilt of 40-50 degrees of the helical axis relative to the membrane normal. Helical structure is also observed for the TM3 peptide in detergent micelles and depends on pH, especially in the C-terminal sequence. In addition, the fluorescence emission of the single tyrosine of the D(E)RY motif in the TM3 peptide exhibits a pronounced pH sensitivity that is abolished when Glu is replaced by Gln, demonstrating that protonation of the conserved Glu side chain affects the structure in the environment of the D(E)RY motif of TM3. The pH regulation of the C-terminal TM3 structure may be an intrinsic feature of the E(D)RY motif in other class I receptors, allowing the coupling of protonation and conformation of membrane-exposed residues in full-length GPCRs.