Bovine Pancreatic Phospholipase A2 Research Articles

High-throughput fluorescence assay for membrane-protein interaction

Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mol% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology domains, an epsin N-terminal homology domain, and a phox homology domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.

A chromogenic substrate for solid-phase detection of phospholipase A2

A method for solid-phase detection of phospholipase A2 (PLA2) was developed. The method uses 1-octanoyloxynaphthalene-3-sulfonic acid, which was found to be a good substrate of PLA2. The substrate is hydrolyzed by PLA2 into 1-naphthol-3-sulfonic acid, which is spontaneously coupled with coexisting diazonium salt to form a red-purple azo dye. Streptomyces and bovine pancreatic PLA2 spotted on a nitrocellulose membrane could be detected by this method with considerable sensitivity. In addition, colonies of recombinant Escherichia coli producing bacterial PLA2 were distinguishable from those producing an inactive mutant PLA2, facilitating high-throughput screening in directed evolution of the enzyme.

ChemInform Abstract: The Inhibitory Mechanism of Phospholipase A2 by Aldehyde Terpenoids

Recently, it has been reported that some unsaturated aldehyde terpenoids inhibit phospholipase A2 (PLA2), which catalyzes hydrolysis of the ester linkage at the sn-2 position of glycerophospholipids. Since the release of arachidonic acid from glycerophospholipids is the rate-limiting step in the production of eicosanoid mediators of inflammation, the inhibitory mechanism of PLA2 by these aldehyde terpenoids has been of biochemical and medicinal interest.It is known at present that the three aldehyde terpenoids, scalaradial, manoalide, and (E) -3methoxycarbonyl-2, 4, 6-trienal compound A strongly inactivate PLA2. As the inhibitory mechanism of PLA2 by manoalide, our group has found that the irreversible modification of Lys 56 in the interfacial recognition site of bovine PLA2 by manoalide is responsible for the inactivation of this enzyme. Although the real reaction mechanism between manoalide and the lysine residues of PLA2 has not been elucidated, our model studies on the reactions between manoalide analogs and mono- and diamines strongly suggested that manoalide reacted with the two close lysine residues to give polymerized compounds. In the case of the (E) -3-methoxycarbonyl-2, 4, 6-trienal compound A, it would inhibit the hydrolytic activity of bovine pancreatic PLA2 by the formation of the dihydropyridine derivatives resulting from the reaction with lysine residues of PLA2via 6π-electrocyclization of the intermediary Schiff bases. Moreover, scalaradial also irreversibly reacts with lysine residues to inactivate PLA2, and the production of the pyrrole derivative has been proposed by Cimino and coworkers based on the model reaction with methylamine.Thus, it would be concluded that the irreversible formation of the product by the reaction with the lysine residue in the interfacial recognition site of PLA2 is essential for sufficient inactivation of PLA2 by unsaturated aldehyde terpenoids such as manoalide, scalaradial, and the (E) -3-methoxycarbonyl2, 4, 6-trienal compounds.

Structural and functional role of water molecules in bovine pancreatic phospholipase A2: a data-mining approach

The water molecules in 25 (21 high-resolution and four atomic resolution) crystal structures of bovine pancreatic phospholipase A(2) have been analyzed in order to identify the invariant water molecules and their possible roles. A total of 24 water molecules have been identified that are invariant in all 25 crystal structures examined. These include the catalytic water molecule, which is directly involved in the enzyme mechanism, and the conserved structural water molecule, which stabilizes the extended hydrogen-bonding network of the active site. Furthermore, many other water molecules stabilize the structure, whilst a few have been found to maintain the active-site geometry and provide coordination to the functionally important calcium ion. The invariant water molecules have been carefully examined and their possible roles in the structure and/or function are discussed. Molecular-dynamics studies of all 25 crystal structures have also been carried out and the results provide a good explanation of and support the findings obtained from the crystal structures.

Structural Biology of Recombinant Bovine Pancreatic Phospholipase A2 and its Inhibitor Complexes

The enzyme phospholipase A2 catalyzes the cleavage of the sn-2 acyl ester bond of phospholipids, leading to the production of free fatty acids and lysophospholipids, which leads to many inflammatory disorders. In view of its pharmaceutical interest, three phospholipase A2 + inhibitor (namely, (i) L-1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine, Transition State Analogue, (ii) 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol, MJ33 and (iii) p-methoxybenzoic acid, anisic acid) complex structures have already been solved and analysed, using the data obtained from X-ray diffraction. These structures provide insight on the mode of binding of the inhibitor molecules at the active site of phospholipase A2. The knowledge of the active site geometry in these inhibitor bound structures, yield valuable information in the design of more useful therapeutic agents. This report reviews only the inhibitor bound recombinant bovine pancreatic phospholipase A2 structures solved using X-ray crystallography.

Phospholipase A2Activates Hemostasis

Background: Phospholipases A2 (PLA2) are aggressive enzymes that can destroy phospholipids of cell membranes. The resulting cell fragments trigger the kallikrein—mediated contact phase of coagulation. The aim of the present study was to expose citrated whole blood to PLA2 and to quantify thrombin generation in recalcified plasma. Methods: Normal citrated blood was exposed to bovine pancreatic or snake PLA2, lipopolysaccharide (LPS), or zymosan A for 30–45 min (RT). After centrifugation the plasma samples were recalcified (10 + 1) with 250 mM CaCl2 in the recalcified coagulation activity assay (RECA). After 0–45 min coagulation reaction time (CRT at 37°C) 1.6 M arginine (final test concentration) was added to stop hemostasis activation and to depolymerize non-crosslinked fibrin. The generated thrombin activity was chromogenically determined. Results: 100 ng/ml bovine pancreatic or snake PLA2 generates about 0.2–0.8 IU/ml thrombin after 15 min CRT. This thrombin generation is similar as that induced by 200 ng/ml LPS or 20 μg/ml zymosan A. Up to 60 ng/ml bovine pancreatic PLA2 the generated thrombin activity is proportional to the PLA2 activity used; 1 μg/ml PLA2 induces much less thrombin, but PLA2 at 10 μg/ml again results into thrombin generation of 0.1–3 IU/ml at 10–15 min CRT. As control, in pooled normal citrated plasma there is no significant change in thrombin generation when exposed to up to 10 μg/ml bovine pancreatic PLA2. Discussion: Elevated plasmatic PLA2 activities (occurring e.g. in trauma, pancreatitis, or sepsis) activate the blood hemostasis system resulting in pathologic disseminated intravascular coagulation (PDIC). It is suggested to diagnose these life threatening states as early as possible, screening all patients for plasmatic thrombin activity.

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 A2

The crystal structures of the monoclinic and trigonal forms of the quadruple mutant K53,56,120,121M of recombinant bovine pancreatic phospholipase A2 (PLA2) have been solved and refined at 1.9 and 1.1 A 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 PLA2 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.

Third calcium ion found in an inhibitor-bound phospholipase A2

The lipolytic enzyme phospholipase A2 plays a crucial role in lipid metabolism and catalyzes hydrolysis of the fatty-acid ester bond at the sn-2 position of phospholipids. Here, the crystal structure (1.7 A resolution) of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 complexed with an organic molecule, p-methoxybenzoic acid (anisic acid), is reported. Residues 60-70 (the surface-loop residues) are ordered and adopt conformations which are different from those normally found in structures in which a second calcium ion is present. It is interesting to note that for the first time a third calcium ion has been identified. In addition, four Tris (2-amino-2-hydroxymethyl-1,3-propanediol) molecules were located. It is believed that one of the Tris molecules plays a role in clamping the third calcium ion and that another is involved in controlling the dynamics of the surface loop through hydrogen bonds.

Atomic resolution (0.97 Å) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2

The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 A). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 A, beta = 101.47 degrees. The structure was solved by molecular replacement and has been refined to a final R factor of 10.6% (Rfree = 13.4%) using 63,926 unique reflections. The final protein model consists of 123 amino-acid residues, two calcium ions, one chloride ion, 243 water molecules and six 2-methyl-2,4-pentanediol molecules. The surface-loop residues 60-70 are ordered and have clear electron density.

Dynamics of ordered water in interfacial enzyme recognition: bovine pancreatic phospholipase A2.

Slippery when wet: Water molecules at the interface between a protein (e.g. bovine pancreatic phospholipase A2) and a micellar substrate act as a lubricant and facilitate movement and binding of the protein at the surface (see picture). Studies of the hydration dynamics before and after complexation reveal that these interactions are crucial for enzymatic function.

Simple intrasequence difference (SID) analysis: an original method to highlight and rank sub-structural interfaces in protein folds. Application to the folds of bovine pancreatic trypsin inhibitor, phospholipase A2, chymotrypsin and carboxypeptidase A.

We present Simple Intrasequence Difference (SID) analysis, a novel bioinformatic technique designed to help comprehend the properties of protein fold topologies. The analysis grades numerically every residue position in a given protein 3D structure according to the topological situation of the position in the folded chain. This results in an expression of the potential contribution of each residue position and its vicinity towards the integrity of the molecular conformation. Contiguous highly graded residues delineate the sub-structural interfaces that arise from the presence within the molecular fold of discrete domains and sub-domains. This comprehensive rendering of the internal arrangement of chain interfacing helps predict the potential for site-specific inductions (e.g. via mutations or ligand binding) of conformational change in the fold. Whereas SID analysis of single folds can convey an idea of the basic potential for topological adjustment in the protein family, comparative SID analysis of related folds focuses attention on those areas of the family fold where evolutionary changes, activation events and ligand binding have had the most topological impact. For demonstration, SID analysis is applied to the folds of pancreatic trypsin inhibitor (Kunitz), phospholipase A(2), chymotrypsin and carboxypeptidase A. We find that many of the potentially vulnerable sub-structural interfaces tend to be protected in the fold interior, in many cases stabilised by disulfide bridges spanning the interface. However, the most prominent interfaces tend to be externally accessible, without remedial stabilisation by disulfide bridges. These latter interfaces are associated so closely with the known functional sites that alterations to the interfacial juxtapositions should influence recognition and catalytic behaviour directly. This shows how side chain mutations, chemical modifications and binding events remote from the sites can nevertheless adjust, via interfacial realignment, the conformations and emergent properties of the sites. The close association also provides clear opportunities for interfacial rearrangements to follow intermolecular recognition events in the sites, facilitating translation of the binding into adjustment of the molecular conformation in areas distant from the sites. As a direct consequence of the topological arrangements, a large proportion of the molecular structure has the capacity to shape the character of the functional sites and, conversely, binding at these sites has the potential to radiate influence to the rest of the molecule. For the enzymes considered, the evidence is consistent with the possibility that primary and secondary binding by the substrate enhances catalytic efficiency by imposing conformational change upon the catalytic centre via adjustments to the fold. This influence may be expressed as favourable adjustment of the catalytic geometry, transition state ensemble, energy propagation pathway, or as a physical strain exerted on the substrate bond to be cleaved. The scale of the adjustments, and their importance to the mechanisms, may have been seriously underestimated.

Changes in flexibility upon binding: Application of the self-consistent pair contact probability method to protein-protein interactions

We extend the self-consistent pair contact probability method to the evaluation of the partition function for a protein complex at thermodynamic equilibrium. Specifically, we adapt the method for multichain models and introduce a parametrization for amino acid-specific pairwise interactions. This method is similar to the Gaussian network model but allows for the adjusting of the strengths of native state contacts. The method is first validated on a high resolution x-ray crystal structure of bovine Pancreatic Phospholipase A2 by comparing calculated B-factors with reported values. We then examine binding-induced changes in flexibility in protein–protein complexes, comparing computed results with those obtained from x-ray crystal structures and molecular dynamics simulations. In particular, we focus on the mouse acetylcholinesterase:fasciculin II and the human α-thrombin:thrombomodulin complexes.

A structural determinant of the unique interfacial binding mode of bovine pancreatic phospholipase A2.

The catalytic steps of the phospholipase A2 (PLA2)-catalyzed hydrolysis of phospholipids are preceded by interfacial binding. Among various pancreatic PLA2s, bovine pancreatic PLA2 (bpPLA2) has a unique interfacial binding mode in which Lys-56 plays an important role in its binding to anionic lipid surfaces. To identify the structural determinant of this unique interfacial binding mode of bpPLA2, we systematically mutated bpPLA2 and measured the effects of mutations on its interfacial binding and activity. First, different cationic clusters were generated in the amino-terminal alpha-helix by the N6R, G7K, and N6R/G7K mutations. These mutations enhanced the binding of bpPLA2 to anionic liposomes up to 15-fold. For these mutants, however, the K56E mutation still caused a large drop in interfacial affinity for and activity toward anionic liposomes, indicating that the generation of a cationic patch in the amino-terminal alpha-helix of bpPLA2 did not change its interfacial binding mode. Second, residues 62-66 that form a part of the pancreatic loop were deleted. For this deletion mutant (Delta62-66), which was as active as wild-type toward anionic liposomes, the K56E and K116E mutations (Delta62-66/K56E and Delta62-66/K116E) did not have significant effects on interfacial affinity. In contrast, the K10E mutation showed a much larger decrease in interfacial affinity (10-fold), indicating the deletion of residues 62-66 caused a major change in the interfacial binding mode. Finally, hydrophobic residues in positions 63 and 65 were replaced by bulkier ones (V63F and V63F/V65L) to pinpoint the structural determinant of the interfacial binding mode of bpPLA2. The effects of K10E and K56E mutations on the interfacial affinity and activity of these mutants showed that Val-63 and Val-65 of bpPLA2 are the structural determinant of its unique interfacial binding mode and that relatively conservative substitutions at these positions result in large changes in the interfacial binding mode among mammalian pancreatic PLA2s. Taken together, this study reveals how minor structural differences among homologous PLA2s can lead to distinct interfacial binding behaviors.

Contributions of residues of pancreatic phospholipase A2 to interfacial binding, catalysis, and activation.

Primary rate and equilibrium parameters for 60 site-directed mutants of bovine pancreatic phospholipase A2 (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 N- and 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 (Kd), the dissociation constant for dissociation of a substrate mimic from the active site of the bound enzyme (KL), and the interfacial Michaelis constants, KM and kcat. Activity is lost (>99.9%) on the substitution of H48 and D49, the catalytic residues. A more than 95% decrease in kcat 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 kcat. Interfacial anionic charge lowers Kd, and induces kcat activation through K56, K53, K119, or K120. Significant changes in KL are seen by the substitution of N6, I9, F22, Y52, K53, N71, Y73, A102, or A103. Changes in KM [=(k2+k-1)/k1] are attributed to kcat (=k2) and KL (=k-1/k1). Some substitutions change more than one parameter, implying an allosteric effect of the binding to the interface on KS, and the effect of the interfacial anionic charge on kcat. 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.

Identification of a coelomic mitogenic factor in Eisenia foetida earthworm

Coelomic fluid of earthworms Eisenia foetida (Oligochaeta, Annelida) exerts a mitogenic activity on murine splenocytes. Total coelomic fluid was subjected to size-exclusion chromatography and a semi-purified mitogenic fraction (fraction 5) was isolated and further characterized. Both coelomic fluid and the semi-purified fraction 5 block concanavalin A (ConA)-induced spleen cell proliferation but exert a synergistic effect on LPS-triggered spleen cell proliferation. Using a polyclonal antiserum neutralizing the mitogenic activity of the semi-purified fraction 5, a 60-kDa component was identified and named CMF (coelomic mitogenic factor). CMF was found to bind ConA which could account for its ability to inhibit ConA-induced spleen cell proliferation. CMF is present in the coelomic fluid as a trimer of a 20-kDa protein. N-terminal amino acid sequence of monomeric CMF reveals partial sequence homology with phospholipase A2 (PLA2). Moreover, CMF-enriched coelomic fluid fraction 5 exerts phospholipase activity comparable with that of bovine pancreatic PLA2. Our results suggest that coelomic fluid of E. foetida contains a ubiquitous PLA2-like enzyme which might be involved in immune reactions in earthworms such as anti-bacterial mechanisms.

アルデヒドテルペノイドによるホスホリパーゼＡ２不活性化機構

Recently, it has been reported that some unsaturated aldehyde terpenoids inhibit phospholipase A2 (PLA2), which catalyzes hydrolysis of the ester linkage at the sn-2 position of glycerophospholipids. Since the release of arachidonic acid from glycerophospholipids is the rate-limiting step in the production of eicosanoid mediators of inflammation, the inhibitory mechanism of PLA2 by these aldehyde terpenoids has been of biochemical and medicinal interest.It is known at present that the three aldehyde terpenoids, scalaradial, manoalide, and (E) -3methoxycarbonyl-2, 4, 6-trienal compound A strongly inactivate PLA2. As the inhibitory mechanism of PLA2 by manoalide, our group has found that the irreversible modification of Lys 56 in the interfacial recognition site of bovine PLA2 by manoalide is responsible for the inactivation of this enzyme. Although the real reaction mechanism between manoalide and the lysine residues of PLA2 has not been elucidated, our model studies on the reactions between manoalide analogs and mono- and diamines strongly suggested that manoalide reacted with the two close lysine residues to give polymerized compounds. In the case of the (E) -3-methoxycarbonyl-2, 4, 6-trienal compound A, it would inhibit the hydrolytic activity of bovine pancreatic PLA2 by the formation of the dihydropyridine derivatives resulting from the reaction with lysine residues of PLA2via 6π-electrocyclization of the intermediary Schiff bases. Moreover, scalaradial also irreversibly reacts with lysine residues to inactivate PLA2, and the production of the pyrrole derivative has been proposed by Cimino and coworkers based on the model reaction with methylamine.Thus, it would be concluded that the irreversible formation of the product by the reaction with the lysine residue in the interfacial recognition site of PLA2 is essential for sufficient inactivation of PLA2 by unsaturated aldehyde terpenoids such as manoalide, scalaradial, and the (E) -3-methoxycarbonyl2, 4, 6-trienal compounds.

High-resolution refinement of orthorhombic bovine pancreatic phospholipase A2.

The X-ray structure of recombinant bovine pancreatic phospholipase A2 (PLA2), which specifically catalyzes the cleavage of the sn-2 acylester bond of phospholipids, has been refined at 1.5 A resolution. The crystal belongs to the space group P212121 with unit-cell parameters a = 47.12, b = 64.59 and c = 38.14 A similar to the native enzyme reported previously by Dijkstra et al. [J. Mol. Biol. (1981), 147, 97-123]. The refinement converged to an R value of 18.4% (Rfree = 22.8%) for 16 374 reflections between 10.0 and 1.5 A resolution. The surface-loop residues (60-70) are ordered in the present orthorhombic recombinant enzyme, but disordered in the trigonal recombinant enzyme. The active-site residues, His48, Asp99, and the catalytic water superimpose well with the trigonal form. Besides the catalytic water which is hydrogen bonded to His48, it is often seen that there is a second water attached to the same N atom of His48 and simultaneously hydrogen bonded to the O atom of Asp49. It is thought that the second water facilitates the tautomerism of His48 for enzyme catalysis. The catalytic water is also hydrogen bonded to the equatorial water coordinated to the calcium ion. In addition to the equatorial water, there is also an axial calcium water and the additional structural water. These five common water molecules are hydrogen bonded to the additional 16 water molecules in the present orthorhombic structure which may further enhance the structural integrity of the active site. Besides the protein and one calcium ion, a total of 134 water molecules were located in the present high-resolution refinement.

1.72 A resolution refinement of the trigonal form of bovine pancreatic phospholipase A2.

The trigonal crystal structure of the recombinant bovine pancreatic phospholipase A2 has been re-refined at a slightly higher resolution (1.72 A). The crystals are trigonal, space group P3121, unit-cell parameters a = b = 46.78 and c = 102.89 A and are isomorphous to the previous structure. The structure was refined to a final crystallographic R value of 19.5% (Rfree = 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 A 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 ( approximately 14 A) 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 water molecules play a role in the activity of the enzyme PLA2. The Ala1 end of the extended active site performs the activation of the phospholid membranes while the opposite end performs the hydrolysis of the monomeric phospholids.

Structure of the complex of bovine pancreatic phospholipase A2 with a transition-state analogue.

The 1.89 A resolution structure of the complex of bovine pancreatic phospholipase A2 (PLA2) with the transition-state analogue L-1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine (TSA) has been determined. The crystal of the complex is trigonal, space group P3121, a = b = 46.58 and c = 102.91 A and isomorphous to the native recombinant wild type (WT). The structure was refined to a final crystallographic R value of 18.0% including 957 protein atoms, 88 water molecules, one calcium ion and all 31 non-H atoms of the inhibitor at 1.89 A resolution. In all, 7 726 reflections [F>2sigma(F)] were used between 8.0 and 1.89 A resolution. The inhibitor is deeply locked into the active-site cleft and coordinates to the calcium ion by displacing the two water molecules in the calcium pentagonal bipyramid by the anionic O atoms of the phosphate and phosphonate group. The hydroxyl group of Tyr69 hydrogen bonds to the second anionic O atom of the phosphate group while that of the phosphonate group replaces the third water, 'catalytic' water, which forms a hydrogen bond to Ndelta1 of His48. The fourth water which also shares Ndelta1 of His48 is displaced by the steric hinderance of the inhibitor. The fifth conserved structural water is still present in the active site and forms a network of hydrogen bonds with the surrounding residues. The structure is compared to the other known TSA-PLA2 complexes.

Molecular Mechanics Calculations of Proteins. Comparison of Different Energy Minimization Strategies

A general strategy for performing energy minimization of proteins using the SYBYL molecular modelling program has been developed. The influence of several variables including energy minimization procedure, solvation, dielectric function and dielectric constant have been investigated in order to develop a general method, which is capable of producing high quality protein structures. Avian pancreatic polypeptide (APP) and bovine pancreatic phospholipase A2 (BP PLA2) were selected for the calculations, because high quality X-ray structures exist and because all classes of secondary structure are represented in the structures. The energy minimized structures were evaluated relative to the corresponding X-ray structures. The overall similarity was checked by calculating RMS distances for all atom positions. Backbone conformation was checked by Ramachandran plots and secondary structure elements evaluated by the length on hydrogen bonds. The dimensions of active site in BP PLA2 is very dependent on electrostatic interactions, due to the presence of the positively charged calcium ion. Thus, the distances between calcium and the calcium-coordinating groups were used as a quality index for this protein. Energy minimized structures of the trimeric PLA2 from Indian cobra (N.n.n. PLA2) were used for assessing the impact of protein-protein interactions. Based on the above mentioned criteria, it could be concluded that using the following conditions: Dielectric constant ϵ = 4 or 20; a distance dependent dielectric function and stepwise energy minimization, it is possible to reproduce X-ray structures very accurately without including explicit solvent molecules.