So Iwata

Structure of the human histamine H1 receptor complex with doxepin.

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 the H1R complex with doxepin, a first-generation H1R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp 4286.48, a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the 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 Lys 1915.39 and/or Lys 179ECL2, both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H1R antagonist specificity against H1R.

The X-ray crystal structures of wild-type and EQ(I-286) mutant cytochrome c oxidases from Rhodobacter sphaeroides.

The structure of cytochrome c oxidase from Rhodobacter sphaeroides has been solved at 2.3/2.8A (anisotropic resolution). This high-resolution structure revealed atomic details of a bacterial terminal oxidase including water molecule positions and a potential oxygen pathway, which has not been reported in other oxidase structures. A comparative study of the wild-type and the EQ(I-286) mutant enzyme revealed structural rearrangements around E(I-286) that could be crucial for proton transfer in this enzyme. In the structure of the mutant enzyme, EQ(I-286), which cannot transfer protons during oxygen reduction, the side-chain of Q(I-286) does not have the hydrogen bond to the carbonyl oxygen of M(I-107) that is seen in the wild-type structure. Furthermore, the Q(I-286) mutant has a different arrangement of water molecules and residues in the vicinity of the Q side-chain. These differences between the structures could reflect conformational changes that take place upon deprotonation of E(I-286) during turnover of the wild-type enzyme, which could be part of the proton-pumping machinery of the enzyme.

Exercise Intolerance Due to Mutations in the Cytochrome b Gene of Mitochondrial DNA

BACKGROUND The mitochondrial myopathies typically affect many organ systems and are associated with mutations in mitochondrial DNA (mtDNA) that are maternally inherited. However, there is also a sporadic form of mitochondrial myopathy in which exercise intolerance is the predominant symptom. We studied the biochemical and molecular characteristics of this sporadic myopathy. METHODS We sequenced the mtDNA cytochrome b gene in blood and muscle specimens from five patients with severe exercise intolerance, lactic acidosis in the resting state (in four patients), and biochemical evidence of complex III deficiency. We compared the clinical and molecular features of these patients with those previously described in four other patients with mutations in the cytochrome b gene. RESULTS We found a total of three different nonsense mutations (G15084A, G15168A, and G15723A), one missense mutation (G14846A), and a 24-bp deletion (from nucleotide 15498 to 15521) in the cytochrome b gene in the five patients. Each of these mutations impairs the enzymatic function of the cytochrome b protein. In these patients and those previously described, the clinical manifestations included progressive exercise intolerance, proximal limb weakness, and in some cases, attacks of myoglobinuria. There was no maternal inheritance and there were no mutations in tissues other than muscle. The absence of these findings suggests that the disorder is due to somatic mutations in myogenic stem cells after germ-layer differentiation. All the point mutations involved the substitution of adenine for guanine, but all were in different locations. CONCLUSIONS The sporadic form of mitochondrial myopathy is associated with somatic mutations in the cytochrome b gene of mtDNA. This myopathy is one cause of the common and often elusive syndrome of exercise intolerance.

Structure and molecular mechanism of a nucleobase-cation- symport-1 family transporter

The nucleobase–cation–symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five 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 transporter LeuTAa and the galactose transporter vSGLT 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 synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport.

The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site.

Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases. These membrane proteins utilize different electron donors through dissimilar access mechanisms. We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase. Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site. A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities. In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase.

Overcoming barriers to membrane protein structure determination.

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ∼30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

Molecular Basis of Alternating Access Membrane Transport by the Sodium-Hydantoin Transporter Mhp1

Triangulating to Mechanism Cellular uptake and release of a variety of substrates are mediated by secondary transporters, but no crystal structures are known for all three fundamental states of the transport cycle, which has limited explanations for their proposed mechanisms. Shimamura et al. (p. 470) report a 3.8-angstrom structure of the inward-facing conformation of the bacterial sodium-benzylhydantoin transport protein, Mhp1, complementing the other two available structures. Molecular modeling for the interconversions of these structures shows a simple rigid body rotation of four helices relative to the rest of the structure in which the protein switches reversibly from outward- to inward-facing. Three complementary crystal structures reveal the mechanism of a transport protein in molecular dynamics simulations. The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens comprises a five-helix inverted repeat, which is widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms 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.

Overcoming the challenges of membrane protein crystallography.

Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet less than 150 unique structures are available. Membrane proteins have proven to be difficult to study owing to their partially hydrophobic surfaces, flexibility and lack of stability. The field is now taking advantage of the high-throughput revolution in structural biology and methods are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins. These technical advances will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future.

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors 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. Here we report the raising of a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A2AAR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes 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 carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β2-adrenergic receptor structure, but locks A2AAR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepTSo, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepTSo represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.