Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations.

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.

Carbon sandwich preparation preserves quality of two-dimensional crystals for cryo-electron microscopy.

Electron crystallography is an important method for determining the structure of membrane proteins. In this paper, we show the impact of a carbon sandwich preparation on the preservation of crystalline sample quality, using characteristic examples of two-dimensional (2D) crystals from gastric H(+),K(+)-ATPase and their analyzed images. Compared with the ordinary single carbon support film preparation, the carbon sandwich preparation dramatically enhanced the resolution of images from flat sheet 2D crystals. As water evaporation is restricted in the carbon-sandwiched specimen, the improvement could be due to the strong protective effect of the retained water against drastic changes in the environment surrounding the specimen, such as dehydration and increased salt concentrations. This protective effect by the carbon sandwich technique helped to maintain the inherent and therefore best crystal conditions for analysis. Together with its strong compensation effect for the image shift due to beam-induced specimen charging, the carbon sandwich technique is a powerful method for preserving crystals of membrane proteins with larger hydrophilic regions, such as H(+),K(+)-ATPase, and thus constitutes an efficient and high-quality method for collecting data for the structural analysis of these types of membrane proteins by electron crystallography.

New crystal structures of PII-type ATPases: excitement continues.

P-type ATPases are ATP-powered ion pumps, classified into five subfamilies (PI-PV). Of these, PII-type ATPases, including Ca2+-ATPase, Na+,K+-ATPase and gastric H+,K+-ATPase, among others, have been the most intensively studied. Best understood structurally and biochemically is Ca2+-ATPase from sarcoplasmic reticulum of fast twitch skeletal muscle (sarco(endo)plasmic reticulum Ca2+-ATPase 1a, SERCA1a). Since publication of the first crystal structure in 2000, it has continuously been a source of excitement, as crystal structures for new reaction intermediates always show large structural changes. Crystal structures now exist for most of the reaction intermediates, almost covering the entire reaction cycle. This year the crystal structure of a missing link, the E1·Mg2+ state, finally appeared, bringing another surprise: bound sarcolipin (SLN). The current status of two other important PII-type ATPases, Na+,K+-ATPase and H+,K+-ATPase, is also briefly described.

Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site.

Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. ATP-driven H(+) uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K(+), resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H(+)/2K(+) to 1H(+)/1K(+) per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K(+) congener (Rb(+)), the 8-Å resolution structure of H(+),K(+)-ATPase in the transition state of dephosphorylation, (Rb(+))E2~AlF, which is distinct from the preceding Rb(+)-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb(+))E2~AlF highly likely represents a single bound Rb(+) ion, which is clearly different from the Rb(+)-free E2AlF or K(+)-bound (K(+))E2~AlF structures. Measurement of radioactive (86)Rb(+) binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb(+) is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H(+)/1K(+)/1ATP transport mode of H(+),K(+)-ATPase, which is a prerequisite for generation of the 10(6)-fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the ß subunit's N terminus and the P domain observed in the (Rb(+))E2~AlF structure, we propose a refined vectorial transport model of H(+),K(+)-ATPase, which must prevail against the highly acidic state of the gastric lumen.

Membrane fusion correlates with surface charge in exocytic vesicles.

Stimulation of gastric parietal cells results in exocytic recruitment of the proton pump (H(+),K(+)-ATPase) from a pool of intracellular membranes (tubulovesicles) to the apical plasma membrane. We have previously reconstituted a step in this process, the homotypic fusion of tubulovesicles, and shown that they also fuse with liposomes in a protein-dependent manner [Duman, J. G., Singh, G., Lee, G. Y., Machen, T. E., and Forte, J. G. (2002) Traffic 3, 203-17]. Further, the lipid composition of the liposomes affects their ability to undergo fusion with tubulovesicles. In the present study, we investigated the lipid requirements for tubulovesicular membrane fusion using a fluorescent probe relaxation assay as well as transfer of protein between tubulovesicles and liposomes of defined composition. Initially, we tested the ability of tubulovesicles to undergo fusion with a panel of synthetic phosphatidylcholine-based liposomes containing a variety of common membrane lipids of various shapes and charges. We found that anionic lipids such as phosphatidylserine, phosphatidic acid, and phosphoinositides were best able to enhance tubulovesicle-liposome fusion and that they did it in a dose-dependent, apparently saturable manner. Next, we altered the lipid compositions of actual tubulovesicles and observed that addition of anionic lipids was able to enhance tubulovesicle-tubulovesicle fusion in vitro; thus, we hypothesized that the charge imparted by the lipids, per se, was responsible for the enhancement of membrane fusion. Accordingly, addition of negative charges to one of two pools of tubulovesicles in a fusion assay using anionic detergents increased membrane fusion; whereas, addition of positively charged cationic detergent decreased membrane fusion and could be used to back-titrate the anionic effects. Surprisingly, when both pools of fusing membranes were loaded with anionic detergents, fusion was markedly increased. The ability of anionic charges to enhance fusion was diminished as the ionic strength of the fusion medium was increased, suggesting that the mechanism of fusion enhancement depends on the surface charge of the membranes. Finally, the fusion reaction was highly dependent on temperature, and anionic charge appears to lower the activation energy of the fusion reaction. Taken together, these data suggest that (1) tubulovesicular fusion is enhanced by an increase in membrane surface negative charge associated with a lower activation energy and (2) neutralization or reversal of the surface charge prevents tubulovesicular fusion.

Correlation between the activities and the oligomeric forms of pig gastric H/K-ATPase.

Membrane-bound H/K-ATPase was solubilized by octaethylene glycol dodecyl ether (C(12)E(8)) or n-octyl glucoside (nOG). H/K-ATPase activity and the distribution of protomeric and oligomeric components were evaluated by high-performance gel chromatography (HPGC) and by single-molecule detection using total internal reflection fluorescence microscopy (TIRFM). As evidenced by HPGC of the C(12)E(8)-solubilized enzyme, the distribution of oligomers was 12% higher oligomeric, 44% diprotomeric, and 44% protomeric species, although solubilization by C(12)E(8) reduced the H/K-ATPase activity to 1.8% of that of the membrane-bound enzyme. The electron microscopic images of the C(12)E(8)-solubilized enzyme showed the presence of protomers and a combination of two and more protomers. While the nOG-solubilized H/K-ATPase retained the same turnover number and 71% of the specific activity as that of the membrane-bound enzyme, 56% higher oligomeric, 34% diprotomeric, and 10% protomeric species were detected. TIRFM analysis of solubilized fluorescein 5'-isothiocyanate (FITC)-modified H/K-ATPase at Lys-518 of the alpha-chain showed a quantized photobleaching of the FITC fluorescence intensity. For the C(12)E(8)-solubilized FITC-enzyme, the fraction of each of the initial relative fluorescence intensity units of 4, 2, and 1 was, respectively, 5%, 44% and 51%. In the case of the nOG-solubilized FITC-enzyme, each fraction of 4 and 2 units was, respectively, 54% and 46% with no detectable 1 unit fraction. This represents the first direct observation of H/K-ATPase in aqueous solution. The correlation between the enzymatic activities and distribution of oligomeric forms of H/K-ATPase by HPGC and the observation of a single molecule of H/K-ATPase and others suggests that the tetraprotomeric form of H/K-ATPase molecules represents the functional species in the membrane.

beta-subunit assembly is essential for the correct packing and the stable membrane insertion of the H,K-ATPase alpha-subunit.

The alpha-subunits of H,K-ATPase (HKAalpha) and Na,K-ATPase require a beta-subunit for maturation. We investigated the role of the beta-subunit in the membrane insertion and stability of the HKAalpha expressed in Xenopus oocytes. Individual membrane segments M1, M2, M3, M4, and M9 linked to a glycosylation reporter act as signal anchor (SA) motifs, and M10 acts as a partial stop transfer motif. In combined HKAalpha constructs, M2 acts as an efficient stop transfer sequence, and M3 acts as a SA sequence. However, M5 and M9 have only partial SA function, and M7 has no SA function. Consistent with the membrane insertion properties of segments in combined alpha constructs, M1-3 alpha-proteins are resistant to cellular degradation, and M1-5 up to M1-10 alpha-proteins are not resistant to cellular degradation. However, co-expression with beta-subunits increases the membrane insertion of M9 in a M1-9 alpha-protein and completely protects M1-10 alpha-proteins against cellular degradation. Our results indicate that HKAalpha N-terminal (M1-M4) membrane insertion and stabilization are mediated by intrinsic molecular characteristics; however, the C-terminal (M5-M10) membrane insertion and thus the stabilization of the entire alpha-subunit depend on intramolecular and intermolecular beta-subunit interactions that are similar but not identical to data obtained for the Na,K-ATPase alpha-subunit.

Three-dimensional structure of the porcine gastric H,K-ATPase from negatively stained crystals.

A low-resolution three-dimensional model of membrane-bound H,K-ATPase from pig gastric mucosa has been reconstructed by electron microscopy and image processing of two-dimensional crystals in negative stain. The crystal formation is induced by magnesium and vanadate, which stabilize the E2 conformation of the enzyme. The unit cell, with a size of a = b = 123 A, gamma = 90 degrees, has tetragonal p4 symmetry. There are four separate alpha beta protomers within each unit cell. The high-contrast region is limited to the cytoplasmic part of the protein. The total volume of the observed asymmetric protein domain corresponds to a molecular mass of 80-90 kDa. It consists mainly of a large pear-shaped domain measuring 60 x 45 A2, with a height of 50 A as measured perpendicular to the membrane plane. A small stalk segment, 20 A in length, forms a connection to the transmembrane region.

Fourier transform infrared spectroscopy study of the secondary structure of the gastric H+,K+-ATPase and of its membrane-associated proteolytic peptides.

Membrane topology of the H+,K+-ATPase has been studied after proteolytic degradation of the protein by proteinase K. Proteinase K had access to either the cytoplasmic part of the protein or to both sides of the membrane. Fourier transform infrared attenuated total reflection spectroscopy indicated that membrane-associated domain of the protein represented about 55% of the native protein, meanwhile the cytoplasmic part represented only 27% of the protein. The secondary structure of the ATPase and of its membrane-associated domains was investigated by infrared spectroscopy. The secondary structure of the membrane-associated structures and of the entire protein was quite similar (alpha-helices, 35%; beta-sheets, 35%; turns, 20%; random, 15%). These data were in agreement with 10 alpha-helical transmembrane segments but suggested a participation of beta-sheet structures in the membrane-associated part of the protein. Polarized infrared spectroscopy indicated that the alpha-helices were oriented nearly perpendicular to the membrane plane. No preferential orientation could be attributed to the beta-sheets. Monitoring the amide hydrogen/deuterium exchange kinetics demonstrated that the membrane associated part of the ATPase molecule is characterized by a relatively high accessibility to the solvent, quite different from that observed for bacteriorhodopsin membrane segments.

The membrane topology of the rat sarcoplasmic and endoplasmic reticulum calcium ATPases by in vitro translation scanning.

The membrane topology of the rat endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) Ca2+ ATPases were investigated using in vitro transcription/translation of fusion vectors containing DNA sequences encoding putative membrane-spanning domains. The sequences of these Ca2+ ATPases are identical except for the COOH-terminal end, which contains an additional predicted transmembrane segment in the ER ATPase. The M0 and M1 fusion vectors (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919) encode the NH2-terminal 101 (M0 vector) or 139 (M1 vector) amino acids of the H,K-ATPase alpha subunit followed by a linker region for insertion of putative transmembrane sequences and, finally, the COOH-terminal 177 amino acids of the H,K-ATPase beta subunit containing five N-linked glycosylation consensus sequences. The linker region was replaced by the putative transmembrane domains of the Ca2+ ATPases, either individually or in pairs. Transcription and translation were performed using [35S]methionine in a reticulocyte lysate system in the absence or presence of canine pancreatic microsomes. The translated fusion protein was identified by autoradiography following separation using SDS-polyacrylamide gel electrophoresis. When testing single transmembrane segments, this method detects signal anchor activity with M0 or stop transfer activity with M1. The first four predicted SERCA transmembrane domains acted as both signal anchor and stop transfer sequences. A construct containing the fifth predicted transmembrane segment was able to act only as a stop transfer sequence. The sixth transmembrane segment did not insert cotranslationally into the membrane. The seventh was able to act as both a signal anchor and stop transfer sequence, and the eighth showed stop transfer ability in the M1 vector. The ninth transmembrane segment had both signal anchor and stop transfer capacity, whereas the tenth transmembrane segment showed only stop transfer sequence properties. The eleventh transmembrane sequence, unique to the ER Ca2+ ATPase, had both signal anchor and stop transfer properties. These translation data provide direct experimental evidence for 8 or 9 of the 10 or 11 predicted transmembrane sequences in the current topological models for the SR or ER Ca2+ ATPases, respectively.