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Abstract: Background Many critical cellular functions are performed by multisubunit circular protein oligomers whose internal geometry has evolved to meet functional requirements. The subunit number is arguably the most critical parameter of a circular protein assembly, affecting the internal and external diameters of the assembly and often impacting on the protein's function. Although accurate structural information has been obtained for several circular proteins, a lack of accurate information on alternative oligomeric states has prevented engineering such transitions. In this study we used the bacterial transcription regulator TRAP as a model system to investigate the features that define the oligomeric state of a circular protein and to question how the subunit number could be manipulated. Methodology/Principal Findings We find that while Bacillus subtilis and Bacillus stearothermophilus TRAP form 11-subunit oligomers, the Bacillus halodurans TRAP exclusively forms 12-subunit assemblies. Significantly, the two states of TRAP are related by a simple rigid body rotation of individual subunits around inter-subunit axes. We tested if such a rotation could be induced by insertion or deletion mutations at the subunit interface. Using wild type 11-subunit TRAP, we demonstrate that removal of five C-terminal residues at the outer side of the inter-subunit axis or extension of an amino acid side chain at the opposite, inner side, increased the subunit number from 11 to 12. Our findings are supported by crystal structures of TRAP oligomers and by native mass spectrometry data. Conclusions/Significance The subunit number of the TRAP oligomer can be manipulated by introducing deletion or addition mutations at the subunit interface. An analysis of available and emerging structural data on alternative oligomeric states indicates that the same principles may also apply to the subunit number of other circular assemblies suggesting that the deletion/addition approach could be used generally to engineer transitions between different oligomeric states.

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Abstract: Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A2A receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A2A selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.

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Abstract: Integral outer membrane proteins (OMPs) play key roles in solute transport, adhesion, and other processes. In Neisseria, they can also function as major protective antigens. Structural, biophysical, and immunological studies of Neisserial OMPs require their isolation in milligram quantities. Purification of any OMP directly from Neisseria would require the growth of large quantities of cell mass, with attendant concerns about safety and convenience. As a result, many investigators have developed methods for expression of OMPs into inclusion bodies in E. coli, followed by refolding of the resolubilized protein. Here we describe such a method, as optimized for the PorA porin but which can be applied, with suitable adaptation, to other OMPs. We also describe an approach to the crystallization of PorA.

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Abstract: In humans, noroviruses (NVs) cause acute epidemic and viral gastroenteritis. NVs do not only infect humans; viruses have also been found in pigs, cows, sheep, mice and dogs. The focus in this project has been on the murine norovirus (MNV). MNV is a member of the viral family Caliciviridae and it consists of a single-stranded, positive sense RNA genome. The genome includes three open reading frames (ORFs), ORF1 encodes for a polyprotein that consists of the precursor to the 6-7 non-structural (NS) proteins. The polyprotein is cleaved by the NS6 protease. The NS6 is responsible for all the cleaving in ORF1 and that makes it an attractive target for antiviral drugs. The NS6 protein structure has been determined at 1.66 Å resolution using X-ray diffraction techniques. Surprisingly, the electron density map revealed density for a peptide bound in the active site. The peptide had a length of 7 residues and originated from the C-terminus of another chain in an adjacent asymmetric unit. The active site triad was composed of the conserved residues; histidine 30, aspargine 54 and cysteine 139, however in the structure the cysteine 139 is mutated to an alanine to inactivate the protease. Activity assays were performed to probe the importance of the residue in position 109 in the β-ribbon located close to the active site. The three full-length constructs with the mutations; I109A, I109S and I109T were found to have less activity than the full-length wt (1-183). A truncated protease, lacking 9 residues in the C-terminus, also had less activity. This indicates that the terminal residues are also important for activity.

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Abstract: 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.