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Bovine heart NADH:ubiquinone oxidoreductase (complex I), which is the largest (about 1 MDa) membrane protein complex in the mitochondrial respiratory chain, catalyzes the electron transfer from NADH to ubiquinone, coupled with proton pumping. We have crystallized bovine complex I in reconstituted lipid bilayers and obtained a three-dimensional density map by the electron crystallographic analysis of a single negatively stained two-dimensional crystal. The asymmetric unit with dimensions of a = 388 Å, b = 129 Å and γ = 90° contains two molecules and is of P1 symmetry. Structural differences between the two molecules indicate flexibility of the hydrophilic domain relative to the membrane-embedded domain.

Received 7 June 2013; accepted 21 June 2013Abstract 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.

Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we present electron crystallographic structures of a mutant human connexin26 (Cx26M34A) and an N-terminal deletion of this mutant (Cx26M34Adel2-7) at 6-Å and 10-Å resolutions, respectively. The three-dimensional map of Cx26M34A was improved by data from 60° tilt images and revealed a breakdown of the hexagonal symmetry in a connexin hemichannel, particularly in the cytoplasmic domain regions at the ends of the transmembrane helices. The Cx26M34A structure contained an asymmetric density in the channel vestibule ("plug") that was decreased in the Cx26M34Adel2-7 structure, indicating that the N terminus significantly contributes to form this plug feature. Functional analysis of the Cx26M34A channels revealed that these channels are predominantly closed, with the residual electrical conductance showing normal voltage gating. N-terminal deletion mutants with and without the M34A mutation showed no electrical activity in paired Xenopus oocytes and significantly decreased dye permeability in HeLa cells. Comparing this closed structure with the recently published X-ray structure of wild-type Cx26, which is proposed to be in an open state, revealed a radial outward shift in the transmembrane helices in the closed state, presumably to accommodate the N-terminal plug occluding the pore. Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration.

Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. In order to characterize the phosphorylation events on H(+),K(+)-ATPase, the properties of fluorinated phosphate analogs [XFs, e.g. aluminum fluoride (AlF), beryllium fluoride (BeF) and magnesium fluoride (MgF)], and the structural differences induced by XFs were investigated. The addition of divalent cations to the XF-inhibited H(+),K(+)-ATPase restores the activity of the AlF- or MgF-inhibited, but not of the BeF-inhibited enzyme, although limited trypsin digestion reveals that they assume the same E(2)P-like state. To clarify the conformational differences induced by XFs, the structure of BeF-bound H(+),K(+)-ATPase was analyzed at 8A resolution. The structure is almost identical to the previously reported AlF-bound E(2)P structure, unlike the distinctive X-ray structure of BeF-bound SERCA, in which the luminal gate was observed to be widely opened. Since the analyzed structure of the H(+),K(+)-ATPase revealed that both AlF and BeF-bound to the P domain were not exposed to the solvent, the dissociation of XFs induced by divalent cations could be interpreted in terms of stability against thermal fluctuations. Furthermore, the conformational differences found between the cytoplasmic domains of H(+),K(+)-ATPase and SERCA provide a framework to understand the characteristic mechanism, by which divalent cations reactivate the XF-inhibited H(+),K(+)-ATPase.

Acid secretion by the stomach results in a pH of about 1. This highly acidic environment is essential for digestion and also acts as a first barrier against bacterial and viral infections. Conversely, too much acid secretion causes gastric ulcer. The mechanism by which this massive proton gradient is generated is of considerable biomedical interest. In this review, we introduce the first molecular model for this remarkable biological phenomenon. The structure of H(+),K(+)-ATPase at 6.5 A resolution was determined by electron crystallography of two-dimensional crystals. The structure shows the catalytic alpha-subunit and the non-catalytic beta-subunit in a pseudo-E(2)P conformation. Different from Na(+),K(+)-ATPase, the N-terminal tail of the beta-subunit is in direct contact with the phosphorylation domain of the alpha-subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the beta-subunit N-terminus allowed the reverse reaction to occur. These results suggest that the N-terminal tail of the beta-subunit functions as a "ratchet", preventing inefficient transport and reverse-flow of protons. We can thus provide a mechanistic explanation for how the H(+),K(+)-ATPase can generate a million-fold proton gradient across the gastric parietal cell membrane, the highest cation gradient known in any mammalian tissue.

Members of the aquaporin (AQP) family are expressed in almost every organism, including 13 homologues in humans. Based on the electron crystallographic structure of AQP1, the hydrogen-bond isolation mechanism was proposed to explain why AQPs are impermeable to protons despite their very fast water conduction. The mechanism by which AQPs exclude protons remained controversial, however. Here we present the structure of AQP4 at 2.8 A resolution obtained by electron crystallography of double-layered two-dimensional crystals. The resolution has been improved from the previous 3.2 A, with accompanying improvement in data quality resulting in the ability to identify individual water molecules. Our structure of AQP4, the predominant water channel in the brain, reveals eight water molecules in the channel. The arrangement of the waters provides support for the hydrogen-bond isolation mechanism. Our AQP4 structure also visualizes five lipids, showing that direct interactions of the extracellular surface of AQP4 with three lipids in the adjoining membrane help stabilize the membrane junction.

The H+,K+-ATPase uses ATP to pump protons across the gastric membrane. We used electron crystallography and limited trypsin proteolysis to study conformational changes in the H+,K+-ATPase. Well-ordered 2D crystals were obtained with detergent-solubilized H+,K+-ATPase at low pH in the absence of nucleotides, E1 state, and in the presence of fluoroaluminate and ADP, mimicking the E1PADP state. Projection maps obtained with frozen-hydrated two-dimensional crystals of the H+,K+-ATPase in these two states looked very similar, suggesting only small conformational changes during the transition from the E1 to the E1P x ADP state. This result differs from the X-ray crystal structures of the related ATPase SERCA, which revealed substantially different conformations in the E1 and E1P x ADP states. To further characterize the conformational changes in the H+,K+-ATPase during its transport cycle, we performed limited proteolysis with trypsin. All examined states of the H+,K+-ATPase, including the E1 and E1P x ADP states present in the 2D crystals,showed characteristic differences in the digestion patterns. While the results from the limited proteolysis experiments thus show that the H+,K+-ATPase adopts distinct conformations during different stages of the transport cycle, the projection maps indicate that the structural rearrangements in the H+,K+-ATPase are much smaller than those observed in the related SERCA ATPase.

In human body, there are thirteen water channels but their expression patterns are tissue specific. Aquaporin-4 (AQP4) is a predominantly expressed water channel in the mammalian brain and an important drug target for treatment of cerebral edema, bipolar disorder, and mesial temporal lobe epilepsy. Recently it was reported that IgG of optic-spinal multiple sclerosis patients bound to AQP4. In order to reveal the function of AQP4, we determined the atomic structure of AQP4 by electron crystallography of double layered two-dimensional crystals. In double layered crystal, each single layered crystal contacts by a short 310 helix in the extracellular loop C. It would suggest that AQP4 shows the weak adhesive activity between adjoining membranes. This is correlated to immunogold labeling of AQP4 in glial lamellae localizing the protein areas where the membranes are separated but also all along junctional regions. Furthermore, from the freeze fracture replica labeling and the mutational experiment, the palmitoylation of N-terminal cysteine residues makes orthogonal array structure unstable on Chinese hamster Ovary (CHO) cell membrane. These findings suggest that there must be the complicated mechanism for control of water content relevant to AQP4 within the brain.

Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. Connexin26 (Cx26) is the second smallest member of the gap junction protein family, and mutations in Cx26 cause certain hereditary human diseases such as skin disorders and hearing loss. Here, we report the electron crystallographic structure of a human Cx26 mutant (M34A). Although crystallization trials used hemichannel preparations, the density map revealed that two hemichannels redocked at their extracellular surfaces into full intercellular channels. These orthorhombic crystals contained two sets of symmetry-related intercellular channels within three lipid bilayers. The 3D map shows a prominent density in the pore of each hemichannel. This density contacts the innermost helices of the surrounding connexin subunits at the bottom of the vestibule. The density map suggests that physical blocking may play an important role that underlies gap junction channel regulation. Our structure allows us to suggest that the two docked hemichannels can be independent and may regulate their activity autonomously with a plug in the vestibule.

Acetazolamide (AZA) reduces the water permeability of aquaporin-4, the predominant water channel in the brain. We determined the structure of aquaporin-4 in the presence of AZA using electron crystallography. Most of the features of the 5-A density map were consistent with those of the previously determined atomic model. The map showed a protruding density from near the extracellular pore entrance, which most likely represents the bound AZA. Molecular docking simulations supported the location of the protrusion as the likely AZA-binding site. These findings suggest that AZA reduces water conduction by obstructing the pathway at the extracellular entrance without inducing a large conformational change in the protein.

Background The mechanisms underlying water transport through aquaporin (AQP) have been debated for two decades. The water permeation phenomenon of AQP seems inexplicable because the Grotthuss mechanism does not allow for simultaneous fast water permeability and inhibition of proton transfer through the hydrogen bonds of water molecules. Scope of review The AQP1 structure determined by electron crystallography provided the first insights into the proton exclusion mechanism despite fast water permeation. Although several studies have provided clues about the mechanism based on the AQP structure, each proposed mechanism remains incomplete. The present review is focused on AQP function and structure solved by electron crystallography in an attempt to fill the gaps between the findings in the absence and presence of lipids. Major conclusions Many AQP structures can be superimposed regardless of the determination method. The AQP fold is preserved even under conditions lacking lipids, but the water arrangement in the channel pore differs. The differences might be explained by dipole moments formed by the two short helices in the lipid bilayer. In addition, structure analyses of double-layered two-dimensional crystals of AQP suggest an array formation and cell adhesive function. General significance Electron crystallography findings not only have contributed to resolve some of the water permeation mechanisms, but have also elucidated the multiple functions of AQPs in the membrane. The roles of AQPs in the brain remain obscure, but their multiple activities might be important in the regulation of brain and other biological functions. This article is part of a Special Issue entitled Aquaporins.

V-ATPases are multisubunit, membrane-bound, energy-converting, cellular machines whose assembly and disassembly is innately connected to their activity in vivo. In vitro V-ATPases show a propensity for disassembly that greatly complicates their functional, and, in particular, structural characterization. Direct structural evidence for early stages of their disassembly has not been reported yet. We analyzed the structure of the V-ATPase from Thermus thermophilus in a single negatively stained two-dimensional (2-D) crystal both by electron tomography and by electron crystallography. Our analysis demonstrated that for 2-D crystals of fragile macromolecular complexes, which are too heterogenous or too few for the merging of image data from many crystals, single-crystal 3-D reconstructions by electron tomography and electron crystallography are expedient tools of analysis. The asymmetric unit in the 2-D crystal lattice contains two different V-ATPase complexes that appear to be in an early stage of disassembly and with either one or both peripheral stalks not being visualized, suggesting the involvement of the peripheral stalks in early stages of disassembly.

In Euglena gracilis, cell membranes on the ridge regions of the striped surface structure are covered with paracrystalline arrays, mainly composed of the integral membrane protein, called IP39. It has recently been cloned as a putative four-membrane-spanning protein with a conserved sequence motif of PMP-22/EMP/MP20/Claudin superfamily. Here, we report the three-dimensional structure of Euglena IP39 at 7 A resolution determined by image-based electron crystallography of two-dimensional (2D) crystals. The 2D crystal array of IP39 appears to be a striated pattern of the antiparallel double-rows, in which the trimeric units of IP39 are longitudinally polymerized, resulting in continuously extending zigzag lines. Each protomer shows an overall cylindrical density (approximately 20 A in diameter) with ‘nock’ and ‘pointed’ shapes at the respective sides of the membrane. Structural analysis of another 2D crystal bound with anti-phosphotyrosine Fab fragments reveals that the ‘nock’-like protruded structure is facing to the cytoplasmic side. A four-helical bundle model is consistently fitted to the EM density map and shows that the transmembrane regions are mainly involved in the intermolecular interactions to form the linear strands. The polymerization pattern of IP39 in the 2D crystal, however, exhibits unexpected interactions between respective protomers. In the trimeric unit of the single strand, one of the three protomers is likely to be rotated at approximately 180 degree in the opposite direction to the others, indicating that there are at least three different ways of possible intermolecular interactions in the transmembrane regions between neighboring protomers. A combination of such multiple interactions would be important for the continuous linear polymerization, thus providing important implications in the strand formation of other four-transmembrane proteins of this family in the lipid environment.

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 E2 P 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 E2 AlF 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 E2 P-stabilizing interaction between the β 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.

Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 A resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 A resolution. The features of the 10-A density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-A map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform.

Acid secretion by the stomach results in a pH of about 1. This highly acidic environment is essential for digestion and also acts as a first barrier against bacterial and viral infections. Conversely, too much acid secretion causes gastric ulcer. The mechanism by which this massive proton gradient is generated is of considerable biomedical interest. In this review, we introduce the first molecular model for this remarkable biological phenomenon. The structure of H(+),K(+)-ATPase at 6.5 A resolution was determined by electron crystallography of two-dimensional crystals. The structure shows the catalytic alpha-subunit and the non-catalytic beta-subunit in a pseudo-E(2)P conformation. Different from Na(+),K(+)-ATPase, the N-terminal tail of the beta-subunit is in direct contact with the phosphorylation domain of the alpha-subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the beta-subunit N-terminus allowed the reverse reaction to occur. These results suggest that the N-terminal tail of the beta-subunit functions as a "ratchet", preventing inefficient transport and reverse-flow of protons. We can thus provide a mechanistic explanation for how the H(+),K(+)-ATPase can generate a million-fold proton gradient across the gastric parietal cell membrane, the highest cation gradient known in any mammalian tissue.

ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) >110 mV, which is composed of a difference in proton concentration (ΔpH) and the electrical potential differences (ΔΨ) across membranes. The optimum rate of synthesis reached 85 s −1 , and the H + /ATP ratio of 4.0 ± 0.1 was obtained. ATP was synthesized at a considerable rate solely by ΔpH, indicating ΔΨ was not absolutely required for synthesis. Consistent with the H + /ATP ratio, cryoelectron micrograph images of 2D crystals of the membrane-bound rotor ring of the V-ATPase at 7.0-Å resolution showed the presence of 12 V o -c subunits, each composed of two transmembrane helices. These results indicate that symmetry mismatch between the rotor and catalytic domains is not obligatory for rotary ATPases/synthases.

H + -ATPase/synthases are membrane-bound rotary nanomotors that are essential for energy conversion in nearly all life forms. A member of the family of the vacuolar-type ATPases (V-ATPases) from Thermus thermophilus , sometimes also termed A-type ATPase, was purified to homogeneity and subjected to two-dimensional (2D) crystallization trials. A novel approach to the 2D crystallization of unstable complexes yielded densely packed sheets of V-ATPase, exhibiting crystalline arrays. Aggregation of the V-ATPase under acidic conditions during reconstitution circumvented the continuous dissociation of the whole complex into the V 1 and V o domains. The resulting three-dimensional aggregates were converted into 2D sheets by the use of a basic buffer, and after a short annealing cycle, ordered arrays of up to 1.5 μm diameter appeared. Fourier transforms calculated from micrographs taken from the negatively stained sample showed diffraction spots to a resolution of 23 A. The Fourier transforms of the untilted images revealed unit-cell dimensions of a = 232 A, b = 132 A, and γ = 90°, and a projection map was calculated by merging 11 images. The most probable molecular packing suggests p22 1 2 1 symmetry of the crystals and dimer contacts between the V 1 domains.

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4–S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures., Journal of Molecular Biology, 425 (22), ISSN:0022-2836, ISSN:1089-8638