Human betacoronaviruses OC43 and HKU1 are endemic respiratory pathogens and, while related, originated from independent zoonotic introductions. OC43 is in fact a host-range variant of the species Betacoronavirus-1, and more closely related to bovine coronavirus (BCoV)-its presumptive ancestor-and porcine hemagglutinating encephalomyelitis virus (PHEV). The β1-coronaviruses (β1CoVs) and HKU1 employ glycan-based receptors carrying 9-Oacetylated sialic acid (9-O-Ac-Sia). Receptor binding is mediated by spike protein S, the main determinant of coronavirus host specificity. For BCoV, a crystal structure for the receptor-binding domain S1 A is available and for HKU1 a cryoelectron microscopy structure of the complete S ectodomain. However, the location of the receptorbinding site (RBS), arguably the single-most important piece of information, is unknown. Here we solved the 3.0-Å crystal structure of PHEV S1 A . We then took a comparative structural analysis approach to map the β1CoV S RBS, using the general design of 9-O-Ac-Siabinding sites as blueprint, backed-up by automated ligand docking, structure-guided mutagenesis of OC43, BCoV, and PHEV S1 A , and infectivity assays with BCoV-S-pseudotyped vesicular stomatitis viruses. The RBS is not exclusive to OC43 and related animal viruses, but is apparently conserved and functional also in HKU1 S1 A . The binding affinity of the HKU1 S RBS toward short sialoglycans is significantly lower than that of OC43, which we attribute to differences in local architecture and accessibility, and which may be indicative for differences between the two viruses in receptor finespecificity. Our findings challenge reports that would map the OC43 RBS elsewhere in S1 A and that of HKU1 in domain S1 B .coronavirus | spike | 9-O-acetylated sialic acid | OC43 | HKU1 C oronaviruses (CoVs; order Nidovirales, family Coronaviridae) are enveloped positive-strand RNA viruses of mammals and birds. So far, four coronaviruses of zoonotic origin are known to have successfully breached the species barrier to become true human pathogens (1-6). These viruses-NL63, 229E, HKU1, and OC43-are persistently maintained in the human population through continuous circulation. Remarkably, the latter two both belong to a single minor clade, "lineage A," in the genus Betacoronavirus. Although generally associated with common colds, HKU1 and OC43 may cause severe and sometimes fatal pulmonary infections in the frail (7, 8), and in rare instances, OC43 may cause lethal encephalitis (9). OC43 and HKU1 are distinct viruses that entered the human population independently to seemingly follow convergent evolutionary trajectories in their adaptation to the novel host (10). OC43 is in fact more related to coronaviruses of ruminants, horses, dogs, rabbits, and swine, with which it has been united in a single species, Betacoronavirus-1.Lineage A betacoronaviruses like HKU1 and OC43 differ from other CoVs in that their virions possess two types of surface projections, both of which are involved in attachment: large 20-nm peplomer...

Background: NAIP5 and NLRC4 induce an innate immune response to intracellular flagellin. Results: Flagellin fragments were identified that induce signaling-competent NAIP5-NLRC4 inflammasomes with 11-and 12-fold symmetry. Conclusion: Conserved flagellin terminal regions induce an inflammasome in which NAIP5 and NLRC4 appear to occupy equivalent positions. Significance: We provide fundamental insights into the formation and structure of hetero-oligomeric inflammasomes.

Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-Å resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domain is displaced by a helix from the preceding domain linker. The two helices conformationally link the scissile-activation peptide and the metal ion-dependent adhesion site required for binding of the ligand C3b. The data suggest that C3b binding displaces the three N-terminal control domains and reshuffles the two central helices.Reshuffling of the helices releases the scissile bond for final proteolytic activation and generates a new interface between the VWA domain and the serine protease domain. This allosteric mechanism is crucial for tight regulation of the complementamplification step in the immune response.Factor B is a tightly regulated, highly specific serine protease. In its activated form, it catalyzes the central amplification step of complement activation to initiate inflammatory responses, cell lysis, phagocytosis and B-cell stimulation 1,2 . Factor B is activated through an assembly process: it binds surface-bound C3b, or its fluid-phase counterpart C3(H 2 O), after which it is cleaved by factor D into fragments Ba (residues 1-234) and Bb (residues 235-739) 3,4 . Fragment Ba dissociates from the complex, leaving behind the alternative pathway C3 convertase complex C3b-Bb, which cleaves C3 into C3a and C3b (see Fig. 1a). This protease complex is intrinsically instable. Once dissociated from the complex, Bb cannot reassociate with C3b 5 . A similar C3 convertase complex is formed upon activation of the classical (antibody-mediated) and lectin-binding pathways, comprised of C4 and C2, which are homologous to C3 and factor B, respectively. The proenzyme factor B consists of three N-terminal complement control protein (CCP) domains, connected by a 45-residue linker to a VWA domain and a C-terminal serine protease (SP) domain, which carries the catalytic center (Fig. 1a). The VWA and SP domains form fragment Bb, and CCP1 through CCP3 and the linker form fragment Ba. Binding of factor B to C3b depends on elements in fragment Ba 6 and the Mg 2+ -dependent metal ion-dependent adhesion site (MIDAS) motif in the VWA domain of fragment Bb 7 . The VWA domain is structurally homologous to inserted (I) domains in integrins. In I domains, ligand binding to the MIDAS is coupled to a B10-Å shift of the a7 activation helix, with concomitant domain rearrangements that activate the integrins 8,9 . Structures of a truncated Bb fragment 10 and its full-length homolog C2a 11 show variable positions of the a7 activation helix affecting the orientation of the VWA and SP domains, which indicates that a related mechanism may occur in convertase formation and dissociation. These structures, however, do not reveal the regulation of the proteolytic activity of factor B. In particular, it is unclear how factor B is maintained in its in...

Insect glutathione-S-transferases (GSTs) are grouped in three classes, I, II and recently III; class I (Delta class) enzymes together with class III members are implicated in conferring resistance to insecticides. Class II (Sigma class) GSTs, however, are poorly characterized and their exact biological function remains elusive. Drosophila glutathione S-transferase-2 (GST-2) (DmGSTS1-1) is a class II enzyme previously found associated specifically with the insect indirect flight muscle. It was recently shown that GST-2 exhibits considerable conjugation activity for 4-hydroxynonenal (4-HNE), a lipid peroxidation product, raising the possibility that it has a major anti-oxidant role in the flight muscle. Here, we report the crystal structure of GST-2 at 1.75 Å resolution. The GST-2 dimer shows the canonical GST fold with glutathione (GSH) ordered in only one of the two binding sites. While the GSH-binding mode is similar to other GST structures, a distinct orientation of helix a6 creates a novel electrophilic substrate-binding site (H-site) topography, largely flat and without a prominent hydrophobic-binding pocket, which characterizes the H-sites of other GSTs. The H-site displays directionality in the distribution of charged/polar and hydrophobic residues creating a binding surface that explains the selectivity for amphipolar peroxidation products, with the polar-binding region formed by residues Y208, Y153 and R145 and the hydrophobic-binding region by residues V57, A59, Y211 and the C-terminal V249. A structure-based model of 4-HNE binding is presented. The model suggest that residues Y208, R145 and possibly Y153 may be key residues involved in catalysis.

Palmitoylation affects membrane partitioning, trafficking and activities of membrane proteins. However, how specificity of palmitoylation and multiple palmitoylations in membrane proteins are determined is not well understood. Here, we profile palmitoylation states of three human claudins, human CD20 and cysteine-engineered prokaryotic KcsA and bacteriorhodopsin by native mass spectrometry. Cysteine scanning of claudin-3, KcsA, and bacteriorhodopsin shows that palmitoylation is independent of a sequence motif. Palmitoylations are observed for cysteines exposed on the protein surface and situated up to 8 Å into the inner leaflet of the membrane. Palmitoylation on multiple sites in claudin-3 and CD20 occurs stochastically, giving rise to a distribution of palmitoylated membrane-protein isoforms. Non-native sites in claudin-3 indicate that membrane-protein function imposed evolutionary restraints on native palmitoylation sites. These results suggest a generic, stochastic membrane-protein palmitoylation process that is determined by the accessibility of palmitoyl-acyl transferases to cysteines on membrane-embedded proteins, and not by a preferred substrate-sequence motif.