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...

No abstract