Multiple binding sites on the pyrin domain of ASC protein allow self-association and interaction with NLRP3 protein.

A key process underlying an innate immune response to pathogens or cellular stress is activation of members of the NOD-like receptor family, such as NLRP3, to assemble caspase-1-activating inflammasome complexes. Activated caspase-1 processes proinflammatory cytokines into active forms that mediate inflammation. Activation of the NLRP3 inflammasome is also associated with common diseases including cardiovascular disease, diabetes, chronic kidney disease, and Alzheimer disease. However, the molecular details of NLRP3 inflammasome assembly are not established. The adaptor protein ASC plays a key role in inflammasome assembly. It is composed of an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain, which are protein interaction domains of the death fold superfamily. ASC interacts with NLRP3 via a homotypic PYD interaction and recruits procaspase-1 via a homotypic caspase recruitment domain interaction. Here we demonstrate that ASC PYD contains two distinct binding sites important for self-association and interaction with NLRP3 and the modulatory protein POP1. Modeling of the homodimeric ASC PYD complex formed via an asymmetric interaction using both sites resembles a type I interaction found in other death fold domain complexes. This interaction mode also permits assembly of ASC PYDs into filaments. Furthermore, a type I binding mode is likely conserved in interactions with NLRP3 and POP1, because residues critical for interaction of ASC PYD are conserved in these PYDs. We also demonstrate that ASC PYD can simultaneously self-associate and interact with NLRP3, rationalizing the model whereby ASC self-association upon recruitment to NLRP3 promotes clustering and activation of procaspase-1.

A structural and catalytic model for zinc phosphoesterases.

A structural model for the active site of phosphoesterases, enzymes that degrade organophosphate neurotoxins, has been synthesised. The ligand [2-((2-hydroxy-3-(((2-hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl)-5-methylbenzyl)(pyridin-2-ylmethyl)amino)acetic acid] (H(3)L1) and two Zn(ii) complexes have been prepared and characterised as [Zn(2)(HL1)(CH(3)COO)](PF(6)).H(2)O and Li[Zn(2)(HL1)](4)(PO(4))(2)(PF(6))(3).(CH(3)OH). The ligand (H(3)L1) and complex [Zn(2)(HL1)(CH(3)COO)](PF(6)).H(2)O were characterised through (1)H NMR, (13)C NMR, mass spectroscopy and microanalysis. The X-ray crystal structure of Li[Zn(2)(HL1)](4)(PO(4))(2)(PF(6))(3).(CH(3)OH) revealed a tetramer of dinuclear complexes, bridged by two phosphate molecules and bifurcating acetic acid arms. Functional studies of the zinc complex with the substrate bis(4-nitrophenyl)phosphate (bNPP) determined the complex with HL1(2-) to be a competent catalyst with k(cat) = 1.26 +/- 0.06 x 10(-6) s(-1).

Cadmium(II) complexes of the glycerophosphodiester-degrading enzyme GpdQ and a biomimetic N,O ligand.

The glycerophosphodiester-degrading enzyme GpdQ from Enterobacter aerogenes is a promising bioremediator owing to its ability to degrade some organophosphate pesticides and diester products originating from the hydrolysis of nerve agents such as VX. Here, the cadmium derivative of GpdQ was prepared by reconstituting the apoenzyme. Catalytic measurements with (Cd(2+))(2)-GpdQ and the phosphodiester substrate bis(4-nitrophenyl)phosphate yield k(cat) = 15 s(-1). The pK(a) of 9.4, determined from the pH dependence of the catalytic activity, implicates a hydroxide ligand as the catalytic nucleophile. Also prepared was the cadmium-containing biomimetic [Cd(2)((HP)(2)B)(OAc)(2)(OH(2))](PF(6)) (where (HP)(2)B is [2,6-bis([(2-pyridylmethyl)(2-hydroxyethyl)amino]methyl)-4-methylphenol]), which mimics the asymmetry of the metal ion coordination in the active site of GpdQ. The phosphoesterase-like activity of [Cd(2)((HP)(2)B)(OAc)(2)(OH(2))](PF(6)) was studied using the substrate bis(2,4-dinitrophenyl)phosphate, yielding a kinetically relevant pK(a) of 8.9, with k(cat) = 0.004 s(-1). In summary, the model is both an adequate structural and a reasonable functional mimic of GpdQ.

Synthesis and characterization of the tetranuclear iron(III) complex of a new asymmetric multidentate ligand. A structural model for purple acid phosphatases.

The ligand, 2-((2-hydroxy-5-methyl-3-((pyridin-2-ylmethylamino)methyl)benzyl)(2-hydroxybenzyl)amino)acetic acid (H(3)HPBA), which contains a donor atom set that mimics that of the active site of purple acid phosphatase is described. Reaction of H(3)HPBA with iron(III) or iron(II) salts results in formation of the tetranuclear complex, [Fe(4)(HPBA)(2)(OAc)(2)(mu-O)(mu-OH)(OH(2))(2)]ClO(4) x 5H(2)O. X-Ray structural analysis reveals the cation consists of four iron(III) ions, two HPBA(3-) ligands, two bridging acetate ligands, a bridging oxide ion and a bridging hydroxide ion. Each binucleating HPBA(3-) ligand coordinates two structurally distinct hexacoordinate iron(III) ions. The two metal ions coordinated to a HPBA(3-) ligand are linked to the two iron(III) metal ions of a second, similar binuclear unit by intramolecular oxide and hydroxide bridging moieties to form a tetramer. The complex has been further characterised by elemental analysis, mass spectrometry, UV-vis and MCD spectroscopy, X-ray crystallography, magnetic susceptibility measurements and variable-temperature Mössbauer spectroscopy.