Suggestive evidence for the involvement of the second calcium and surface loop in interfacial binding: monoclinic and trigonal crystal structures of a quadruple mutant of phospholipase A 2 (original) (raw)
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Journal of Molecular Biology, 2002
Phospholipase A 2 catalyses hydrolysis of the ester bond at the C2 position of 3-sn-phosphoglycerides. Here we report the 1.9 Å resolution crystal structure of the triple mutant K56,120,121M of bovine pancreatic phospholipase A 2 . The structure was solved by molecular replacement method using the orthorhombic form of the recombinant phospholipase A 2 . The final protein model contains all the 123 amino acid residues, two calcium ions, 125 water molecules and one 2-methyl-2-4-pentanediol molecule. The model has been refined to a crystallographic R-factor of 19.6% (R free of 25.9%) for all data between 14.2 Å and 1.9 Å . The residues 62 -66, which are in a surface loop, are always disordered in the structures of bovine pancreatic phospholipase A 2 and its mutants. It is interesting to note that the residues 62 -66 in the present structure is ordered and the conformation varies substantially from those in the previously published structures of this enzyme. An unexpected and interesting observation in the present structure is that, in addition to the functionally important calcium ion in the active site, one more calcium ion is found near the N terminus. Detailed structural analyses suggest that binding of the second calcium ion could be responsible for the conformational change and the ordering of the surface loop. Furthermore, the results suggest a structural reciprocity between the k cat p allosteric site and surface loop at the i-face, which represents a newly identified structural property of secreted phospholipase A 2 .
Crystal Structures of the Free and Anisic Acid Bound Triple Mutant of Phospholipase A2
Journal of Molecular Biology, 2003
Phospholipase A 2 catalyses the hydrolysis of the ester bond of 3-sn-phosphoglycerides. Here, we report the crystal structures of the free and anisic acid-bound triple mutant (K53,56,120M) of bovine pancreatic phospholipase A 2 . In the bound triple mutant structure, the small organic molecule p-anisic acid is found in the active site, and one of the carboxylate oxygen atoms is coordinated to the functionally important primary calcium ion. The other carboxylate oxygen atom is hydrogen bonded to the phenolic hydroxyl group of Tyr69. In addition, the bound anisic acid molecule replaces one of the functionally important water molecules in the active site. The residues 60 -70, which are in a loop (surface loop), are disordered in most of the bovine pancreatic phospholipase A 2 structures. It is interesting to note that these residues are ordered in the bound triple mutant structure but are disordered in the free triple mutant structure. The organic crystallization ingredient 2-methyl-2,4-pentanediol is found near the active site of the free triple mutant structure. The overall tertiary folding and stereochemical parameters for the final models of the free and anisic acid-bound triple mutant are virtually identical.
Journal of Molecular Biology, 1997
Activation of phospholipase A 2 (PLA 2 ) upon binding to phospholipid assemblies is poorly understood. X-ray crystallography revealed little structural change in the enzyme upon binding of monomeric substrate analogs, whereas small conformational changes in PLA 2 complexed with substrate micelles and an inhibitor were found by NMR. The structure of PLA 2 bound to phospholipid bilayers is not known. Here we uncover by FTIR spectroscopy a splitting in the a-helical region of the amide I absorbance band of PLA 2 upon binding to lipid bilayers. We provide evidence that a higher frequency component, which is only observed in the membrane-bound enzyme, is a property of more¯exible helices. Formation of exible helices upon interaction with the membrane is likely to contribute to PLA 2 activation.
Biochemistry, 1999
Primary rate and equilibrium parameters for 60 site-directed mutants of bovine pancreatic phospholipase A 2 (PLA2) are analyzed so incremental contributions of the substitution of specific residues can be evaluated. The magnitude of the change is evaluated so a functional role in the context of the Nand C-domains of PLA2 can be assigned, and their relationship to the catalytic residues and to the i-face that makes contact with the interface. The effect of substitutions and interfacial charge is characterized by the equilibrium dissociation constant for dissociation of the bound enzyme from the interface (K d), the dissociation constant for dissociation of a substrate mimic from the active site of the bound enzyme (K L *), and the interfacial Michaelis constants, K M * and k* cat. Activity is lost (>99.9%) on the substitution of H48 and D49, the catalytic residues. A more than 95% decrease in k* cat is seen with the substitution of F5, I9, D99, A102, or F106, which form the substrate binding pocket. Certain residues, which are not part of the catalytic site or the substrate binding pocket, also modulate k* cat. Interfacial anionic charge lowers K d , and induces k* cat activation through K56, K53, K119, or K120. Significant changes in K L * are seen by the substitution of N6, I9, F22, Y52, K53, N71, Y73, A102, or A103. Changes in K M * [)(k 2 +k-1)/ k 1 ] are attributed to k* cat ()k 2) and K L * ()k-1 /k 1). Some substitutions change more than one parameter, implying an allosteric effect of the binding to the interface on K S *, and the effect of the interfacial anionic charge on k* cat. Interpreted in the context of the overall structure, results provide insights into the role of segments and domains in the microscopic events of catalytic turnover and processivity, and their allosteric regulation. We suggest that the interfacial recognition region (i-face) of PLA2, due to the plasticity of certain segments and domains, exercises an allosteric control on the substrate binding and chemical step.
1.72 Å Resolution Refinement of the Trigonal Form of Bovine Pancreatic Phospholipase A2
Acta Crystallographica Section D Biological Crystallography, 1998
The trigonal crystal structure of the recombinant bovine pancreatic phospholipase A2 has been re-refined at a slightly higher resolution (1.72 Å). The crystals are trigonal, space group P3121, unit-cell parameters a = b = 46.78 and c = 102.89 Å and are isomorphous to the previous structure. The structure was refined to a final crystallographic R value of 19.5% (R free = 28.4%) using 10 531 reflections. A total of 106 solvent molecules were included in the refinement compared with the earlier refinement which contains only 85 water molecules and 8 925 reflections at 1.8 Å resolution. The root-mean-square deviation from the ideal bond lengths and bond angles is considerably better in the present refinement. The active site is extended (\sim14 Å) from Ala1 to the calcium. The three catalytic residues (Asp99, His48 and the catalytic water) are connected by the conserved structural water and the N-terminal Ala1 on one side, and by the calcium through an equatorial water on the other. The...
J Mol Biol, 1998
The amino-terminal, 138 amino acid C2 domain of cytosolic phospholipase A 2 (cPLA 2-C2) mediates an initial step in the production of lipid mediators of in¯ammation: the Ca 2-dependent translocation of the enzyme to intracellular membranes with subsequent liberation of arachidonic acid. The high resolution solution structure of this Ca 2-dependent, lipid-binding domain (CaLB) has been determined using heteronuclear three-dimensional NMR spectroscopy. Secondary structure analysis, derived from several sets of spectroscopic data, shows that the domain is composed of eight antiparallel b-strands with six interconnecting loops that ®ts the``type II'' topology for C2 domains. Using a total of 2370 distance and torsional restraints, the structure was found to be a b-sandwich in the``Greek key'' motif. The solution structure of cPLA 2-C2 domain is very similar to the X-ray crystal structure of the C2 domain of phospholipase-C-d and phylogenetic analysis clari®es the structural role of highly conserved residues. Calorimetric studies further demonstrate that cPLA 2-C2 binds two Ca 2 with observed K d s of approximately 2 mM in an entropically assisted process. Moreover, regions on cPLA 2-C2 interacting with membranes were identi®ed by 15 N-HSQC-spectroscopy of cPLA 2-C2 in the presence of low molecular weight lipid micelles. An extended binding site was identi®ed that binds the phosphocholine headgroup in a Ca 2-dependent manner and also interacts with proximal regions of the membrane surface. Based upon these results, a structural model is presented for the mechanism of association of cPLA 2 with its membrane substrate.
Acta Crystallographica Section D Biological Crystallography, 1999
Crystal structures of the active-site mutants D99A and H48Q and the calcium-loop mutant D49E of bovine phospholipase A2 have been determined at around 1.9 Å resolution. The D99A mutant is isomorphous to the orthorhombic recombinant enzyme, space group P212121. The H48Q and the calcium-loop mutant D49E are isomorphous to the trigonal recombinant enzyme, space group P3121. The two active-site mutants show no major structural perturbations. The structural water is absent in D99A and, therefore, the hydrogen-bonding scheme is changed. In H48Q, the catalytic water is present and hydrogen bonded to Gln48 N, but the second water found in native His48 is absent. In the calcium-loop mutant D49E, the two water molecules forming the pentagonal bipyramid around calcium are absent and only one O atom of the Glu49 carboxylate group is coordinated to calcium, resulting in only four ligands.
Journal of Biological Chemistry, 1998
The Ca 2؉-dependent lipid binding domain of the 85-kDa cytosolic phospholipase A 2 (cPLA 2) is a homolog of C2 domains present in protein kinase C, synaptotagmin, and numerous other proteins involved in signal transduction. NH 2-terminal fragments of cPLA 2 spanning the C2 domain were expressed as inclusion bodies in Escherichia coli, extracted with solvent to remove phospholipids, and refolded to yield a domain capable of binding phospholipid vesicles in a Ca 2؉-dependent manner. Unlike other C2 domains characterized to date, the cPLA 2 C2 domain bound preferentially to vesicles comprised of phosphatidylcholine in response to physiological concentrations of Ca 2؉. Binding of the cPLA 2 C2 domain to vesicles in the presence of excess Ca 2؉ chelator was induced by high concentrations of salts that promote hydrophobic interactions. Despite the selective hydrolysis of arachidonyl-containing phospholipid vesicles by cPLA 2 , the cPLA 2 C2 domain did not discriminate among phospholipid vesicles containing saturated or unsaturated sn-2 fatty acyl chains. Moreover, the cPLA 2 C2 domain bound to phospholipid vesicles containing sn-1 and-2 ether linkages and sphingomyelin at Ca 2؉ concentrations that caused binding to vesicles containing ester linkages, demonstrating that the carbonyl oxygens of the sn-1 and-2 ester linkage are not critical for binding. These results suggest that the cPLA 2 C2 domain interacts primarily with the headgroup of the phospholipid. The cPLA 2 C2 domain displayed selectivity among group IIA cations, preferring Ca 2؉ approximately 50fold over Sr 2؉ and nearly 10,000-fold over Ba 2؉ for vesicle binding. No binding to vesicles was observed in the presence of greater than 10 mM Mg 2؉. Such strong selectivity for Ca 2؉ over Mg 2؉ reinforces the view that C2 domains link second messenger Ca 2؉ to signal transduction events at the membrane. Much of the interest in the 85-kDa cytosolic PLA 2 (cPLA 2) 1