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Biomedical subjects

M Allaire

Publications and source records attributed to M Allaire.

12 recordsLinked to original sources
PubMed2001

A viral phospholipase A2 is required for parvovirus infectivity.

Sequence analysis revealed phospholipase A2 (PLA2) motifs in capsid proteins of parvoviruses. Although PLA2 activity is not known to exist in viruses, putative PLA2s from divergent parvoviruses, human B19, porcine parvovirus, and insect GmDNV (densovirus from Galleria mellonella), can emulate catalytic properties of secreted PLA2. Mutations of critical amino acids strongly reduce both PLA2 activity and, proportionally, viral infectivity, but cell surface attachment, entry, and endocytosis by PLA2-deficient virions are not affected. PLA2 activity is critical for efficient transfer of the viral genome from late endosomes/lysosomes to the nucleus to initiate replication. These findings offer the prospect of developing PLA2 inhibitors as a new class of antiviral drugs against parvovirus infections and associated diseases.

Amino Acid Motifs↗
PubMed2000

Channeling efficiency in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase domain: the effects of site-directed mutagenesis of NADP binding residues.

The three-dimensional structure of the dehydrogenase-cyclohydrolase bifunctional domain of the human trifunctional enzyme indicates that Arg-173 and Ser-197 are within 3 A of the 2'-phosphate of bound NADP. Site-directed mutagenesis confirms that Arg-173 is essential for efficient binding and cannot be substituted by lysine. R173A and R173K have detectable dehydrogenase activity, but the K(m) values for NADP are increased by at least 500-fold. The S197A mutant has a K(m) for NADP that is only 20-fold higher than wild-type, indicating that it plays a supporting role. Forward and reverse cyclohydrolase activities of all the mutants were unchanged, except that the reverse cyclohydrolase activity of mutants that bind NADP poorly, or lack Ser-197, cannot be stimulated by 2',5'-ADP. The 50% channeling efficiency in the forward direction is not improved by the addition of exogenous NADPH and cannot be explained by premature dissociation of the dinucleotide from the ternary complex. As well, channeling is unaffected in mutants that exhibit a wide range of dinucleotide binding. Given that dinucleotide binding is unrelated to substrate channeling efficiency in the D/C domain, we propose that the difference in forward and reverse channeling efficiencies can be explained solely by the movement of the methenylH(4)folate between two overlapping subsites to which it has different binding affinities.

Aminohydrolases↗
PubMed1998

Crystallization and preliminary analysis of chondroitinase AC from Flavobacterium heparinum.

Chondroitinase AC (E.C. 4.2.2.5) overexpressed in its host, Flavobacterium heparinum, was crystallized by vapor diffusion using polyethylene glycol methyl ether as precipitant. It crystallizes in the space group P43212 or its enantiomorph with a = b = 87.1 and c = 193.1 A and one molecule in the asymmetric unit. Crystals diffract to a maximum of 2.5 A resolution on a rotating-anode source. Screening for heavy-atom derivatives identified a lead compound that binds to a single site on the protein. Further screening is in progress.

Binding Sites↗
PubMed1998

The 3-D structure of a folate-dependent dehydrogenase/cyclohydrolase bifunctional enzyme at 1.5 A resolution.

BACKGROUND: The interconversion of two major folate one-carbon donors occurs through the sequential activities of NAD(P)-dependent methylene[H4]folate dehydrogenase (D) and methenyl[H4]folate cyclohydrolase (C). These activities often coexist as part of a multifunctional enzyme and there are several lines of evidence suggesting that their substrates bind at overlapping sites. Little is known, however, about the nature of this site or the identity of the active-site residues for this enzyme family. RESULTS: We have determined, to 1.5 A resolution, the structure of a dimer of the D/C domain of the human trifunctional cytosolic enzyme with bound NADP cofactor, using the MAD technique. The D/C subunit is composed of two alpha/beta domains that assemble to form a wide cleft. The cleft walls are lined with highly conserved residues and NADP is bound along one wall. The NADP-binding domain has a Rossmann fold, characterized by a modified diphosphate-binding loop fingerprint-GXSXXXG. Dimerization occurs by antiparallel interaction of two NADP-binding domains. Superposition of the two subunits indicates domain motion occurs about a well-defined hinge region. CONCLUSIONS: Analysis of the structure suggests strongly that folate-binding sites for both activities are within the cleft, providing direct support for the proposed overlapping site model. The orientation of the nicotinamide ring suggests that in the dehydrogenase-catalyzed reaction hydride transfer occurs to the pro-R side of the ring. The identity of the cyclohydrolase active site is not obvious. We propose that a conserved motif-Tyr52-X-X-X-Lys56- and/or a Ser49-Gln100-Pro102 triplet have a role in this activity.

Aminohydrolases↗
PubMed1996

Crystallization of the bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase domain of the human trifunctional enzyme.

Methylenetetrahydrofolate([H4] folate) dehydrogenase (D) and methenyl[H4] folate cyclohydrolase (C) coexist as a bifunctional enzyme (DC) or as the amino-terminal domain of a trifunctional enzyme (DCS) where the third activity is 10-formyl[H4]folate synthetase (S). Two crystal forms of the DC domain of the human cytosolic DCS enzyme have been grown from polyethyleneglycol solution. The monoclinic P2(1) crystals diffract to 2.8 A with a = 72.5 A, b = 68.5 A, c = 125.2 A, and beta = 91.8 degrees but were found to be twinned. The orthorhombic P2(1)2(1)2(1) crystals diffract to 2.5 A with a = 67.7 A, b = 135.9 A, c = 61.6 A, and contain two molecules per asymmetric unit.

Aminohydrolases↗
PubMed1994

Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases.

The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 A resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an intermolecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

3C Viral Proteases↗
PubMed1993

Hepatitis A virus 3C proteinase: some properties, crystallization and preliminary crystallographic characterization.

Several isoforms of the wild-type and three mutant hepatitis A virus (HAV) 3C proteinases have been isolated and characterized. The active site cysteine residue (residue 172) was found to be responsible for the formation of some of these isoforms. The double mutant C24S/C172A of the HAV 3C proteinase, in which both cysteine residues have been replaced by site-directed mutagenesis, was crystallized. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph, P6(5)22) with unit cell dimensions a = b = 65.2 A, c = 246.1 A and diffract X-rays to 2.3 A resolution.

3C Viral Proteases↗
PubMed1990

Inactivation of mammalian fructose diphosphate aldolases by COOH terminus autophosphorylation.

Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.

Amino Acids↗
PubMed1988

Sequential radiation damage in protein crystallography.

Radiation damage in protein crystals is described in terms of a sequential process of protein disordering. A new radiation-damage model has been tested against data from several protein crystals and can describe radiation damage corresponding to loss of the original intensity in excess of 80%. The model is an extension of previous models which characterize radiation damage in terms of successive conformational transitions of the protein from an undamaged to a spatially disordered to finally an amorphous state. The proposed model provides a more-general positional characterization of the disordered protein and includes, prior to the disordered state, a new dose-dependent state in which the protein conformation resembles the undamaged protein. Comparison of this model with the best previous model shows that the proposed model provides an improved fit to radiation-damage data.

Adenosine Triphosphatases↗
PubMed1987

Molecular architecture of rabbit skeletal muscle aldolase at 2.7-A resolution.

The molecular architecture of the rabbit skeletal muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) tetramer has been determined to 2.7-A resolution. Solution of the three-dimensional structure of rabbit muscle aldolase utilized phase information from a single isomorphous Pt(CN)4(2-) derivative, which was combined with iterative-phase refinement based upon the noncrystallographic 222-fold symmetry exhibited by the tetramer subunits. The electron-density map calculated from the refined phases (mf = 0.72) was interpreted on the basis of the known amino acid sequence (363 amino acids per subunit). The molecular architecture of the aldolase subunit corresponds to a singly wound beta-barrel of the parallel alpha/beta class structures as has been observed in triose phosphate isomerase, pyruvate kinase, phosphogluconate aldolase, as well as others. Close contacts between tetramer subunits are virtually all between regions of hydrophobic residues. Contrary to other beta-barrel structures, the known active-site residues are located in the center of the beta-barrel and are accessible to substrate from the COOH side of the beta-barrel. Biochemical and crystallographic data suggest that the COOH-terminal region of aldolase covers the active-site pocket from the COOH side of the beta-barrel and mediates access to the active site. On the basis of sequence studies, active-site residues as well as residues lining the active-site pocket have been totally conserved throughout evolution. By comparison, homology in the COOH-terminal region is minimal. It is suggested that the amino acid sequence of the COOH-terminal region may be, in part, the basis for the variable specific activities aldolases exhibit toward their substrates.

Animals↗