The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.
five helix inverted repeat superfamily; hydantoin; membrane transport; Mhp1; molecular recognition; nucleobase-cation-symport, NCS1, family
The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. These receptors regulate glucose and fatty acid metabolism, thereby ameliorating type 2 diabetes. The full-length human AdipoR1 and a series of N-terminally truncated mutants of human AdipoR1 and AdipoR2 were expressed in insect cells. In small-scale size exclusion chromatography, the truncated mutants AdipoR1Δ88 (residues 89–375) and AdipoR2Δ99 (residues 100–386) eluted mostly in the intact monodisperse state, while the others eluted primarily as aggregates. However, gel filtration chromatography of the large-scale preparation of the tag-affinity-purified AdipoR1Δ88 revealed the presence of an excessive amount of the aggregated state over the intact state. Since aggregation due to contaminating nucleic acids may have occurred during the sample concentration step, anion-exchange column chromatography was performed immediately after affinity chromatography, to separate the intact AdipoR1Δ88 from the aggregating species. The separated intact AdipoR1Δ88 did not undergo further aggregation, and was successfully purified to homogeneity by gel filtration chromatography. The purified AdipoR1Δ88 and AdipoR2Δ99 proteins were characterized by thermostability assays with 7-diethylamino-3-(4-maleimidophenyl)-4-methyl coumarin, thin layer chromatography of bound lipids, and surface plasmon resonance analysis of ligand binding, demonstrating their structural integrities. The AdipoR1Δ88 and AdipoR2Δ99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method. X-ray diffraction data sets were obtained at resolutions of 2.8 and 2.4 Å, respectively.
Membrane protein; Adiponectin receptors AdipoR1 and AdipoR2; Purification; Antibody; Crystallization; Lipidic mesophase
CAAX proteins play essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis 1. The ~120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases 2. Proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of their C-terminal CAAX motifs 3 (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve cysteine prenylation, -AAX tripeptide cleavage, and methylation of the carboxyl prenylated Cys residue. The major CAAX protease activity is mediated by the Ras and a-factor converting enzyme 1 (Rce1), an integral membrane protease of the endoplasmic reticulum 4,5. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here, we report the crystal structure of a Methanococcus maripaludis homolog of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site, are distinct from other intramembrane proteases (IMPs). Catalytic residues are located ~10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. The farnesyl lipid is proposed to bind to a site at the opening of two transmembrane α-helices, which then positions the scissile bond adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.
Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as, hypertension, heart failure and epilepsy and they are well-established drug targets2. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli1,3, where both EM and crystal structures are available4-6. NhaA is made up of two distinct domains, a Core domain and a Dimerisation domain. In the NhaA crystal structure a cavity is located between the two domains providing access to the ion-binding site from the inward-facing surface of the protein1,4. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, where a conformational change is thought to occur7. To date, the only reported NhaA crystal structure is of the low pH inactivated form4. Here, we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the Core and Dimerisation domains are in different positions to those seen in NhaA and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to directly coordinate ion-binding1,8,9, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the Core domain, some 20° against the Dimerisation interface. We conclude that despite their fast transport rates of up to 1500 ions/sec3, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.
Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble functional domain V1 (A3B3DF) and an integral membrane domain Vo (ac), where V1 and Vo domains are connected by a central stalk, composed of D-, F-, and d-subunits; and two peripheral stalks (E- and G-subunits). We identified 120 interacting residues of A3B3 heterohexamer with D-subunit in DF heterodimer in the crystal structures of A3B3 and A3B3DF. In our previous study, we reported 10 mutants of E. hirae V1-ATPase, which showed lower binding affinities of DF with A3B3 complex leading to higher initial specific ATPase activities compared to the wild-type. In this study, we identified a mutation of A-subunit (LV476-7AA) at its C-terminal domain resulting in the A3B3 complex with higher binding affinities for wild-type or mutant DF heterodimers and lower initial ATPase activities compared to the wild-type A3B3 complex, consistent with our previous proposal of reciprocal relationship between the ATPase activity and the protein-protein binding affinity of DF axis to the A3B3 catalytic domain of E. hirae V-ATPase. These observations suggest that the binding of DF axis at the contact region of A3B3 rotary ring is relevant to its rotation activity.
Site-directed mutation; Reconstitution; Catalytic domain; ATPase assay; Surface plasmon resonance; Enterococcus hirae
Vacuolar ATPases (V-ATPases) function as proton pumps in various cellular membrane systems. The hydrophilic V1 portion of the V-ATPase is a rotary motor, in which a central-axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex by using ATP hydrolysis energy. We have previously reported crystal structures of Enterococcushirae V-ATPase A3B3 and A3B3DF (V1) complexes; the result suggested that the DF axis induces structural changes in the A3B3 complex through extensive protein-protein interactions. In this study, we mutated 10 residues at the interface between A3B3 and DF complexes and examined the ATPase activities of the mutated V1 complexes as well as the binding affinities between the mutated A3B3 and DF complexes. Surprisingly, several V1 mutants showed higher initial ATPase activities than wild-type V1-ATPase, whereas these mutated A3B3 and DF complexes showed decreased binding affinities for each other. However, the high ATP hydrolysis activities of the mutants decreased faster over time than the activity of the wild-type V1 complex, suggesting that the mutants were unstable in the reaction because the mutant A3B3 and DF complexes bound each other more weakly. These findings suggest that strong interaction between the DF complex and A3B3 complex lowers ATPase activity, but also that the tight binding is responsible for the stable ATPase activity of the complex.
A systematic approach to the scaling and merging of data from multiple crystals in macromolecular crystallography is introduced and explained.
The availability of intense microbeam macromolecular crystallography beamlines at third-generation synchrotron sources has enabled data collection and structure solution from microcrystals of <10 µm in size. The increased likelihood of severe radiation damage where microcrystals or particularly sensitive crystals are used forces crystallographers to acquire large numbers of data sets from many crystals of the same protein structure. The associated analysis and merging of multi-crystal data is currently a manual and time-consuming step. Here, a computer program, BLEND, that has been written to assist with and automate many of the steps in this process is described. It is demonstrated how BLEND has successfully been used in the solution of a novel membrane protein.
clustering; multiple crystals; BLEND; scaling; merging; multi-crystal data sets
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of Vo, a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21−3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1NT, and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1NT); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.
In this study, the glucansucrase from the dental caries pathogen S. mutans was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant.
Glucansucrases encoded by Streptococcus mutans play essential roles in the synthesis of sticky dental plaques. Based on amino-acid sequence similarity, glucansucrases are classified as members of glycoside hydrolase family 70 (GH 70). Data on the crystal structure of GH 70 glucansucrases have yet to be reported. Here, the GH 70 glucansucrase GTF-SI from S. mutans was overexpressed in Escherichia coli strain BL21 (DE3), purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Orthorhombic GTF-SI crystals belonging to space group P21212 were obtained. A diffraction data set was collected to 2.1 Å resolution.
glucansucrase; dental caries; Streptococcus mutans
G protein-coupled receptors (GPCRs) are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states.1-13 Conformational changes induced by extracellular ligands binding to GPCRs result in a cellular response through the activation of G-proteins. The A2A adenosine receptor (A2AAR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain.14 In this study, we have successfully raised a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket. The structure of the A2AAR-antibody Fab fragment (Fab2838) complex reveals that the fragment, unexpectedly, recognises the intracellular surface of A2AAR and that its complementarity determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein C-terminal fragment in the active opsin structure1 and to the CDR-3 of the nanobody in the active β2 adrenergic receptor structure2 but locks the A2AAR in an inactive conformation. These results shed light on a novel strategy to modulate GPCR activity.
The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine-H1 receptor (H1R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of H1R complex with doxepin, a first-generation H1R-antagonist. Doxepin sits deep in the ligand binding pocket and directly interacts with the highly conserved Trp4286.48, a key residue in GPCR activation. This well-conserved pocket with mostly hydrophobic nature contributes to low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H1R-antagonists reveals that the unique carboxyl-group present in this class of compounds interacts with Lys1915.39 and/or Lys179ECL2, both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors defining how minor differences in receptor lead to pronounced selectivity differences with small molecules.
Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital.
We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6–7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials.
We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.
G-protein coupled receptor; Membrane protein; High expression; Screening; Receptor variants; Structural study; Saccharomyces cerevisiae
Alternating access mechanism in the POT family of oligopeptide transporters
Proton-dependent oligopeptide transporters are required for the uptake of diet-derived peptides in all kingdoms of life. The crystal structure of a bacterial transporter in the inward open conformation, together with a published structure in an occluded conformation, reveals the peptide transport mechanism.
Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.
alternating access mechanism; major facilitator superfamily; peptide transport; PepT1; POT family
High cholesterol levels greatly increase the risk of cardiovascular disease. By its conversion into bile acids, about 50% of cholesterol is eliminated from the body. However bile acids released from the bile duct are constantly recycled, being reabsorbed in the intestine via the Apical Sodium dependent Bile acid Transporter (ASBT). It has been shown in animal models that plasma cholesterol levels are significantly lowered by specific inhibitors of ASBT1,2, thus ASBT is a target for hypercholesterolemia drugs. Here, we describe the crystal structure of a bacterial homologue of ASBT from Neisseria meningitidis (ASBTNM) at 2.2Å. ASBTNM contains two inverted structural repeats of five transmembrane helices. A Core domain of six helices harbours two sodium ions while the remaining helices form a Panel-like domain. Overall the architecture of the protein is remarkably similar to the sodium-proton antiporter NhaA3 despite no detectable sequence homology. A bile acid molecule is situated between the Core and Panel domains in a large hydrophobic cavity. Residues near to this cavity have been shown to affect the binding of specific inhibitors of human ASBT4. The position of the bile acid together with the molecular architecture suggests the rudiments of a possible transport mechanism.
Sulfur is an essential component for the biosynthesis of the sulfur-containing amino acids l-methionine and l-cysteine. The crystal structure of the periplasmic aliphatic sulfonate-binding protein SsuA from E. coli has been solved at 1.75 Å resolution in a substrate-free state. Comparison with the substrate-bound protein shows significant movement of one of the two domains.
Sulfur is an essential component for the biosynthesis of the sulfur-containing amino acids l-methionine and l-cysteine. Under sulfur-starvation conditions, bacteria are capable of scavenging sulfur from sulfur-containing compounds and transporting it across membranes. Here, the crystal structure of the periplasmic aliphatic sulfonate-binding protein SsuA from Escherichia coli is reported at 1.75 Å resolution in the substrate-free state. The overall structure of SsuA resembles the structures of other periplasmic binding proteins and contains two globular domains that form a cleft. Comparison with other periplasmic binding proteins revealed that one of the domains has been displaced by a rigid movement of 17°. Interestingly, the tight crystal packing appears to be mediated by a 13-amino-acid tail from the cloning that folds within the cleft of the next monomer.
periplasmic binding proteins; aliphatic sulfonates; substrate-free state; extended C-terminus tail; intermediate open state
Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.
The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.
Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.
Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield high-resolution structures.
► Benchmarked the stability required for crystallization in small sized detergents ► Membrane protein stability is inherent to the protein rather than detergent specific ► Membrane proteins stable in LDAO are more likely to yield well-diffracting crystals ► Eukaryotic membrane proteins are 3-fold less stable in 12M than prokaryotic proteins
Mhp1, a hydantoin transporter from M. liquefaciens, was purified and crystallized. Diffraction data were collected to 2.85 Å resolution; the crystal belonged to the orthorhombic space group P212121.
The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P212121, unit-cell parameters a = 79.7, b = 101.1, c = 113.8 Å). A complete data set has been collected from a single crystal to a resolution of 2.85 Å with 64 741 independent observations (94% complete) and an R
merge of 0.12. Further experimental phasing methods are under way.
transporters; nucleobase:cation symporter 1 family; membrane proteins; hydantoins
Crystal structures of a membrane protein transporter in three different conformational states provide insights into the transport mechanism.
Secondary active transporters move molecules across cell membranes by coupling this process to the energetically favourable downhill movement of ions or protons along an electrochemical gradient. They function by the alternating access model of transport in which, through conformational changes, the substrate binding site alternately faces either side of the membrane. Owing to the difficulties in obtaining the crystal structure of a single transporter in different conformational states, relatively little structural information is known to explain how this process occurs. Here, the structure of the sodium-benzylhydantoin transporter, Mhp1, from Microbacterium liquefaciens, has been determined in three conformational states; from this a mechanism is proposed for switching from the outward-facing open conformation through an occluded structure to the inward-facing open state.
membrane transport; transport protein; alternating access; hydantoins
The structure of the sodium-benzylhydantoin transport protein, Mhp1, from Microbacterium liquefaciens comprises a 5-helix inverted repeat, which is widespread amongst secondary transporters. Here we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 Å resolution, complementing its previously-described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward- facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8 and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.
Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone coupled reduction of polysulfide, a process important in extreme environments such as deep-sea vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4 Å resolution, revealing how the PsrA subunit recognizes and reduces its unique poly anionic substrate. The integral membrane subunit PsrC was characterized using the natural substrate menaquinone-7 and inhibitors, providing a comprehensive representation of a quinone binding site and revealing the presence of a water filled cavity connecting the quinone binding site on the periplasmic side to the cytoplasm. These results suggest that polysulfide reductase could be a key energy-conserving enzyme of the T. thermophilus respiratory chain, utilizing polysulfide as the terminal electron acceptor and pumping protons across the membrane via a previously unknown mechanism.
Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on > 40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either β-sheets or less commonly from α-helices. The most common architecture is the β-barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallisation can be made. Herein we analyse the crystallisation data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallisation is presented based on the present analysis.
Outer Membrane Proteins; Protein Crystallisation; Membrane protein crystallisation; Screen design
The ‘Nucleobase-Cation-Symport-1’, NCS1, transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85 Å resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, ten of which are arranged in two inverted repeats of 5 helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine (LeuTAa) and the galactose (vSGLT) transporters reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronised by the inverted repeat helices 3 and 8, providing the structural basis of the ‘alternating access’ model for membrane transport.
Vacuolar-type ATPases (V-ATPases) exist in various cellular membranes of many organisms to regulate physiological processes by controlling the acidic environment. Here, we have determined the crystal structure of the A3B3 subcomplex of V-ATPase at 2.8 Å resolution. The overall construction of the A3B3 subcomplex is significantly different from that of the α3β3 sub-domain in FoF1-ATP synthase, because of the presence of a protruding ‘bulge' domain feature in the catalytic A subunits. The A3B3 subcomplex structure provides the first molecular insight at the catalytic and non-catalytic interfaces, which was not possible in the structures of the separate subunits alone. Specifically, in the non-catalytic interface, the B subunit seems to be incapable of binding ATP, which is a marked difference from the situation indicated by the structure of the FoF1-ATP synthase. In the catalytic interface, our mutational analysis, on the basis of the A3B3 structure, has highlighted the presence of a cluster composed of key hydrophobic residues, which are essential for ATP hydrolysis by V-ATPases.
crystal structure; FoF1; proton pump; rotary motor; V-ATPase
It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for high-resolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 μGFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.