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.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombincleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors. (Blood. 2008;112: 2803-2809) Introduction TAFI 1,2 is a pro-metallocarboxypeptidase that links the coagulation and fibrinolytic systems. TAFI is activated by thrombin, the thrombin-thrombomodulin complex or plasmin. 3 Activated TAFI (TAFIa) inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine residues from partially degraded fibrin that are required for positive feedback in tissue plasminogen-activator dependent plasmin generation. In addition, TAFIa has been implicated in modulation of the inflammatory response by inactivating bradykinin and the anaphylatoxins C3a and C5a. 4,5 Although it is a powerful antifibrinolytic agent, there are no known physiologic inhibitors of TAFIa. Instead, the half-life of TAFIa activity is regulated by its intrinsic instability. The inactivation rate, 5 to 10 minutes at 37°C, is highly temperature-dependent, suggesting that inactivation involves a large conformational change. 6 This is also suggested by the susceptibility of the inactive enzyme, TAFIai to proteolytic cleavage by thrombin at Arg302, a site that is cryptic in TAFI and TAFIa. 6,7 The stability of TAFIa is an important determinant for its antifibrinolytic potential because TAFIa inhibits fibrinolysis through a threshold-dependent mechanism. [8][9][10] Full-length TAFI consists of 401 amino acids divided into 2 domains: the first 92 amino acids form the activation peptide; the next 309 amino acids form the catalytic domain. The activation peptide restricts substrate access to the catalytic cleft in the zymogen. TAFI is activated through cleavage at Arg92, which releases the activation peptide.TAFI is highly homologous to the pancreatic procarboxypeptidases with 42% sequence identity to human procarboxypeptidase B (pro...

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis X binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam 2 CSK 4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam 2 CSK 4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.S. aureus | Toll-like receptor | immune evasion | innate immunity | crystal structure

Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation that NapG and H, but not NapF, are essential for electron transfer from ubiquinol to NapAB. The decreased yield of biomass resulting from loss of NapF in a Ubi + Men+ strain implicates NapF in an energyconserving role coupled to the oxidation of ubiquinol. We propose that NapG and H form an energyconserving quinol dehydrogenase functioning as either components of a proton pump or in a Q cycle, as electrons are transferred from ubiquinol to NapC. IntroductionEnergy-conserving electron transfer pathways in enteric bacteria are usually depicted as a series of substratespecific dehydrogenases feeding electrons into a common quinone pool, from which they are transferred via specific quinol dehydrogenases to cytochrome oxidases during aerobic growth or terminal reductases during anaerobic growth. This is clearly an oversimplification, however, because there are three types of functional quinone in Escherichia coli, ubiquinone 8 (UQ) and the naphthoquinones demethylmenaquinone (DMK) and menaquinone (MK). UQ is generally regarded as the 'aerobic' quinone in the sense that ubiquinone is far more abundant than MK and DMK during aerobic growth (Wallace and Young, 1977;Wissenbach et al., 1992;Soballe and Poole, 1999). Furthermore, UQ is essential for succinoxidase activity. Conversely, the naphthoquinone pool is essential for anaerobic respiration using nitrite, fumarate, dimethyl sulphoxide (DMSO) or trimethylamine N-oxide (TMAO) (Wissenbach et al., 1990;1992;Tyson et al., 1997). The selectivity of quinones for specific electron donors or acceptors can be explained by the difference in mid-point redox potential between the UQ/UQH 2 couple (E m,7 = +113 mV) and the MK/MKH 2 couple (E m,7 = -74 mV) (Soballe and Poole, 1999). In addition, there may also be structural constraints that limit the enzymes of the respiratory chain to binding a specific quinone.Nitrate respiration in E. coli has a unique position as electrons from both UQH 2 and MKH 2 , but not DMKH 2 , can be used for nitrate reduction (Wissenbach et al., 1990;1992;Tyson et al., 1997). E. coli expresses three nitrate reductases. Two of them, nitrate reductases A and Z, are membrane bound and reduce nitrate in the cytoplasm. SummaryThe nap operon of Escherichia coli K-12, encoding a periplasmic nitrate reductase (Nap), encodes seven proteins. The catalytic complex in the periplasm, NapA-NapB, is assumed to receive electrons from the quinol pool via the membrane-bound cytochrome NapC. Like NapA, B and C, a fourth polypeptide, NapD, is also essential for Nap activity. However, none of the remaining three polypeptides, NapF, G and H, which are predicted to encode non-haem, iron-sulphur proteins, are essential for Nap activity, and their function is currently unknown. The relative rates of growth and electron transfer from physiological substrates to Nap have been investigated using strains defective in the two membrane-bound nitrate reductases, and als...

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Nap (periplasmic nitrate reductase) operons of many bacteria include four common, essential components, napD, napA, napB and napC (or a homologue of napC ). In Escherichia coli there are three additional genes, napF, napG and napH, none of which are essential for Nap activity. We now show that deletion of either napG or napH almost abolished Nap-dependent nitrate reduction by strains defective in naphthoquinone synthesis. The residual rate of nitrate reduction (approx. 1% of that of napG+ H+ strains) is sufficient to replace fumarate reduction in a redox-balancing role during growth by glucose fermentation. Western blotting combined with beta-galactosidase and alkaline phosphatase fusion experiments established that NapH is an integral membrane protein with four transmembrane helices. Both the N- and C-termini as well as the two non-haem iron-sulphur centres are located in the cytoplasm. An N-terminal twin arginine motif was shown to be essential for NapG function, consistent with the expectation that NapG is secreted into the periplasm by the twin arginine translocation pathway. A bacterial two-hybrid system was used to show that NapH interacts, presumably on the cytoplasmic side of, or within, the membrane, with NapC. As expected for a periplasmic protein, no NapG interactions with NapC or NapH were detected in the cytoplasm. An in vitro quinol dehydrogenase assay was developed to show that both NapG and NapH are essential for rapid electron transfer from menadiol to the terminal NapAB complex. These new in vivo and in vitro results establish that NapG and NapH form a quinol dehydrogenase that couples electron transfer from the high midpoint redox potential ubiquinone-ubiquinol couple via NapC and NapB to NapA.

Fibrillar collagens, the most abundant proteins in the vertebrate body, are involved in a plethora of biological interactions. Plasma protein von Willebrand factor (VWF) mediates adhesion of blood platelets to fibrillar collagen types I, II, and III, which is essential for normal haemostasis. High affinity VWF-binding sequences have been identified in the homotrimeric collagen types II and III, however, it is unclear how VWF recognizes the heterotrimeric collagen type I, the superstructure of which is unknown. Here we present the crystal structure of VWF domain A3 bound to a collagen type III-derived homotrimeric peptide. Our structure reveals that VWF-A3 interacts with all three collagen chains and binds through conformational selection to a sequence that is one triplet longer than was previously appreciated from platelet and VWF binding studies. The VWF-binding site overlaps those of SPARC (also known as osteonectin) and discodin domain receptor 2, but is more extended and shifted toward the collagen amino terminus. The observed collagen-binding mode of VWF-A3 provides direct structural constraints on collagen I chain registry. A VWF-binding site can be generated from the sequences RGQAGVMF, present in the two α1 (I) chains, and RGEOGNIGF, in the unique α2(I) chain, provided that α2(I) is in the middle or trailing position. Combining these data with previous structural data on integrin binding to collagen yields strong support for the trailing position of the α2(I) chain, shedding light on the fundamental and long-standing question of the collagen I chain registry.X-ray crystallography | extracellular matrix V on Willebrand factor (VWF) mediates blood platelet adhesion to sites of vascular damage and is essential for platelet adhesion under conditions of high shear stress. Defective processing and/or mutations in VWF are associated with various haemostatic disorders such as haemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and von Willebrand disease, which is the most common genetic bleeding disorder in humans (1). The mature VWF protein consists of 2,050 amino acid monomers that are disulfide-linked into multimers ranging from 0.5 to 10 MDa in size with the larger multimers being more active in hemostasis. VWF multimer size is regulated by ADAMTS13 through proteolytic cleavage within the VWF A2 domain. Upon vascular damage, the VWF A3 domain interacts with exposed collagens I and III. Subsequent transient interactions between the VWF A1 domain and Glycoprotein Ibα (GPIbα) on the surface of blood platelets reduces platelet velocity to allow subsequent stable platelet adhesion and activation, which ultimately results in thrombus deposition.The crystal structure of the collagen-binding VWF A3 domain has been solved (2-4) and the collagen-binding site within the A3 domain has been mapped in extensive site-directed mutagenesis studies and by NMR (5-7). However, detailed structural information on the VWF-collagen interaction has thus far been lacking.Collagens, the most abundant proteins in mammalian...

Leukocyte-associated immunoglobulinlike receptor-1 (LAIR-1), one of the most widely spread immune receptors, attenuates immune cell activation when bound to specific sites in collagen. The collagenbinding domain of LAIR-1 is homologous to that of glycoprotein VI (GPVI), a collagen receptor crucial for platelet activation. Because LAIR-1 and GPVI also display overlapping collagen-binding specificities, a common structural basis for collagen recognition would appear likely. Therefore, it is crucial to gain insight into the molecular interaction of both receptors with their ligand to prevent unwanted cross-reactions during therapeutic intervention. We determined the crystal structure of LAIR-1 and mapped its collagen-binding site by nuclear magnetic resonance (NMR) titrations and mutagenesis. Our data identify R59, E61, and W109 as key residues for collagen interaction. These residues are strictly conserved in LAIR-1 and GPVI alike; however, they are located outside the previously proposed GPVI collagenbinding site. Our data provide evidence for an unanticipated mechanism of collagen recognition common to LAIR-1 and GPVI. This fundamental insight will contribute to the exploration of specific means of intervention in collagen-induced signaling in immunity and hemostasis. (Blood.