Interfacial Recognition by Bee Venom Phospholipase A2: Insights into Nonelectrostatic Molecular Determinants by Charge Reversal Mutagenesis (original) (raw)
Related papers
Biochemistry, 1998
The basis for tight binding of bee venom phospholipase A 2 (bvPLA2) to anionic versus zwitterionic phospholipid interfaces is explored by charge reversal mutagenesis of basic residues (lysines/ arginines to glutamates) on the putative membrane binding surface. Single-site mutants and, surprisingly, multisite mutants (2-5 of the 6 basic residues mutated) are fully functional on anionic vesicles. Mutants bind tightly to anionic vesicles, and active-site substrate and Ca 2+ binding are not impaired. Multisite mutants undergo intervesicle exchange slightly faster than wild type, especially in the presence of salt. It is estimated that electrostatic contribution to interfacial binding is modest, perhaps 2-3 kcal/mol of the estimated 15 kcal/mol. Elution properties of bvPLA2 from HPLC columns containing solid phases of tightly packed monolayers of phosphocholine amphiphiles suggest that ionic effects provide a modest portion of the interfacial binding energy and that this contribution decreases as the number of cationic residues mutated is increased. These results are consistent with the observation that Gila monster venom PLA2 (Pa2), which is homologous to bvPLA2, has high activity on anionic vesicles despite the fact that it has only a single basic residue on its putative interfacial recognition face. Results with bvPLA2 mutants show that manoalogue and 12-epi-scalaradial inactivate bvPLA2 by modification of K94. Also, deletion of the large -loop (residues 99-118) is without consequence for interfacial binding and catalysis of bvPLA2. All together, the preferential binding of bvPLA2 to anionic vesicles versus phosphatidylcholine vesicles is mainly due to factors other than electrostatics. Therefore hydrogen-bonding and hydrophobic interactions must provide a major portion of the interfacial binding energy, and this is consistent with recent spectroscopic studies.
Mapping the Interfacial Binding Surface of Human Secretory Group IIa Phospholipase A2
Biochemistry, 1997
Human secretory group IIa phospholipase A 2 (hIIa-PLA 2) contains a large number of prominent cationic patches on its molecular surface and has exceptionally high affinity for anionic surfaces, including anionic membranes. To identify the cationic amino acid residues that support binding of hIIa-PLA 2 to anionic membranes, we have performed extensive site-directed mutagenesis of this protein and measured vesicle binding and interfacial kinetic properties of the mutants using polymerized liposomes and nonpolymerized anionic vesicles. Unlike other secretory PLA 2 s, which have a few cationic residues that support binding of enzyme to anionic membranes, interfacial binding of hIIa-PLA 2 is driven in part by electrostatic interactions involving a number of cationic residues forming patches on the putative interfacial binding surface. Among these residues, the amino-terminal patch composed of Arg-7, Lys-10, and Lys-16 makes the most significant contribution to interfacial adsorption, and this is supplemented by contributions from other patches, most notably Lys-74/Lys-87/Arg-92 and Lys-124/Arg-127. For these mutants, complete vesicle binding occurs in the presence of high vesicle concentrations, and under these conditions the mutants display specific activities comparable to that of wild-type enzyme. These studies indicate that electrostatic interactions between surface lysine and arginine residues and the interface contribute to interfacial binding of hIIa-PLA 2 to anionic vesicles and that cationic residues closest to the opening of the active-site slot make the most important interactions with the membrane. However, because the wild type binds extremely tightly to anionic vesicles, it was not possible to exactly determine what fraction of the total interfacial binding energy is due to electrostatics.
Acta Crystallographica Section D Biological Crystallography, 2006
The crystal structures of the monoclinic and trigonal forms of the quadruple mutant K53,56,120,121M of recombinant bovine pancreatic phospholipase A 2 (PLA 2 ) have been solved and refined at 1.9 and 1.1 Å resolution, respectively. Interestingly, the monoclinic form reveals the presence of the second calcium ion. Furthermore, the surface-loop residues are ordered and the conformation of residues 62-66 is similar to that observed in other structures containing the second calcium ion. On the other hand, in the trigonal form the surface loop is disordered and the second calcium is absent. Docking studies suggest that the second calcium and residues Lys62 and Asp66 from the surface loop could be involved in the interaction with the polar head group of the membrane phospholipid. It is hypothesized that the two structures of the quadruple mutant, monoclinic and trigonal, represent the conformations of PLA 2 at the lipid interface and in solution, respectively. A docked structure with a phospholipid molecule and with a transition-state analogue bound, one at the active site coordinating to the catalytic calcium and the other at the second calcium site, but both at the i-face, is presented.
Biophysical Journal, 2003
Phospholipase A 2 (PLA 2 ) binds to membranes and catalyzes phospholipid hydrolysis, thus initiating the biosynthesis of lipid-derived mediators of inflammation. A snake-venom PLA 2 was completely inhibited by covalent modification of the catalytic histidine 48 by p-bromophenacyl bromide. Moreover, His 48 modification affected PLA 2 structure, its membranebinding affinity, and the effects of PLA 2 on the membrane structure. The native PLA 2 increased the order parameter of fluid membranes, whereas the opposite effect was observed for gel-state membranes. The data suggest membrane dehydration by PLA 2 and the formation of PLA 2 -membrane hydrogen bonding. The inhibited PLA 2 had lower membrane-binding affinity and exerted weaker effects on membrane hydration and on the lipid-order parameter. Although membrane binding resulted in formation of more flexible a-helices in the native PLA 2 , which corresponds to faster amide hydrogen exchange, the modified enzyme was more resistant to hydrogen exchange and experienced little structural change upon membrane binding. The data suggest that 1), modification of a catalytic residue of PLA 2 induces conformational changes that propagate to the membranebinding surface through an allosteric mechanism; 2), the native PLA 2 acquires more dynamic properties during interfacial activation via membrane binding; and 3), the global conformation of the inhibited PLA 2 , including the a-helices, is less stable and is not influenced by membrane binding. These findings provide further evidence for an allosteric coupling between the membrane-binding (regulatory) site and the catalytic center of PLA 2 , which contributes to the interfacial activation of the enzyme.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1978
We examined the action of porcine pancreatic and bee-venom phospholipase A2 towards bilayers of phosphatidylcholine as a function of several physical characteristics of the lipid-water interface. 1. Unsonicated liposomes of dimyristoyl phosphatidylcholine are degraded by both phospholipases in the temperature region of the phase transition only (cf. Op den Kamp et al. (1974) Biochim. Biophys. Acta 345, 253--256 and Op den Kamp et al. (1975) Biochim. Biophys. Acta 406, 169--177). With sonicates the temperature range in which hydrolysis occurs is much wider. This discrepancy between liposomes and sonicates cannot be ascribed entirely to differences in available substrate surface. 2. Below the phase-transition temperature the phospholipases degrade dimyristoyl phosphatidylcholine single-bilayer vesicles with a strongly curved surface much more effectively than larger single-bilayer vesicles with a relatively low degree of curvature. 3. Vesicles composed of egg phosphatidylcholine can be degraded by pancreatic phospholipase A2 at 37 degrees C, provided that the substrate bilayer is strongly curved. The bee-venom enzyme shows a similar, but less pronounced, preference for small substrate vesicles. 4. In a limited temperature region just above the transition temperature of the substrate the action of both phospholipases initially proceeds with a gradually increasing velocity. This stimulation is presumably due to an increase of the transition temperature, effectuated by the products of the phospholipase action. 5. Structural defects in the substrate bilayer, introduced by sonication below the phase-transition temperature (cf. Lawaczeck et al. (1976) Biochim. Biophys. Acta 443, 313--330) facilitate the action of both phospholipases. The results lead to the general conclusion that structural irregularities in the packing of the substrate molecules facilitate the action of phospholipases A2 on phosphatidylcholine bilayers. Within the phase transition and with bilayers containing structural defects these irregularities represent boundaries between separate lipid domains. The stimulatory effect of strong bilayer curvature can be ascribed to an overall perturbation of the lipid packing as well as to a change in the phase-transition temperature.
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.
Interaction of phospholipase A2 and phospholipid bilayers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1982
Binding of phospholipase A 2 from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)-rac-glycero-3-phosphocholine (diether-PCt4) is studied in the presence and absence of 1-tetradecanoyl-sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC 14 alone. In contrast, the porcine enzyme binds only to ternary codispersions of diaikyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of the pig-pancreatic enzyme to vesicles of the diether-PC t4 could not be detected even after long incubation (up to 24 h) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies.
Journal of Biological Chemistry, 2002
Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A(2) (sPLA(2)s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA(2)s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 microm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA(2)s, with Me-Indoxam being the most generally potent sPLA(2) inhibitor. A dramatic correlation exists between the ability of the sPLA(2)s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA(2)s are uniquely efficient in this regard.
Biochemistry, 2004
Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A 2 (PLA2) to form the premicellar complexes (E i # ) are characterized to discern the short-range specific interactions. Typically, E i # complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (E i # , i ) 1, 2, 3). In the presence of calcium, the dissociation constant K 1 for the E 1 # complex of PLA2 with decyl sulfate (CMC ) 4500 µM) is 70 µM with a Hill coefficient n 1 ) 2.1 ( 0.2; K 2 for E 2 # is 750 µM with n 2 ) 8 ( 1, and K 3 for E 3 # is 4000 µM with an n 3 value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of E i # ; (c) K i and n i do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the E i # complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in E i # . The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of E i # . We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E* and E i # aggregates is discussed.