共查询到20条相似文献,搜索用时 0 毫秒
1.
Poi MJ Tomaszewski JW Yuan C Dunlap CA Andersen NH Gelb MH Tsai MD 《Journal of molecular biology》2003,329(5):997-1009
This work describes in-depth NMR characterization of a unique low-barrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A(2) (sPLA(2), from bee venom and bovine pancreas) and a transition-state analog inhibitor HK32. A downfield proton NMR resonance, at 17-18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48. These results led to a hypothesis that the downfield resonance represents the proton (H(epsilon 2) of H48) involved in the H-bonding between D99 and H48, in analogy with serine proteases. However, this was shown not to be the case by use of the bovine enzyme labeled with specific [15N(epsilon 2)]His. Instead, the downfield resonance arises from H(delta1) of H48, which forms a hydrogen bond with a non-bridging phosphonate oxygen of the inhibitor. Further studies showed that this proton displays a fractionation factor of 0.62(+/-0.06), and an exchange rate protection factor of >100 at 285 K and >40 at 298 K, which are characteristic of a LBHB. The pK(a) of the imidazole ring of H48 was shown to be shifted from 5.7 for the free enzyme to an apparent value of 9.0 in the presence of the inhibitor. These properties are very similar to those of the Asp em leader His LBHBs in serine proteases. Possible structural bases and functional consequences for the different locations of the LBHB between these two types of enzymes are discussed. The results also underscore the importance of using specific isotope labeling, rather than extrapolation of NMR results from other enzyme systems, to assign the downfield proton resonance to a specific hydrogen bond. Although our studies did not permit the strength of the LBHB to be accurately measured, the data do not provide support for an unusually strong hydrogen bond strength (i.e. >10 kcal/mol). 相似文献
2.
Alpha-lipoic acid (ALA) and its reduced form dihydrolipoic acid (DHLA) are powerful antioxidants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. The mechanism of anti-inflammatory activity of ALA and DHALA is not known. The present study describes the interaction of ALA and DHALA with pro-inflammatory secretory PLA(2) enzymes from inflammatory fluids and snake venoms. In vitro enzymatic inhibition of sPLA(2) from Vipera russellii, Naja naja and partially purified sPLA(2) enzymes from human ascitic fluid (HAF), human pleural fluid (HPF) and normal human serum (HS) by ALA and DHLA was studied using (14)C-oleate labeled Escherichia coli as the substrate. Biophysical interaction of ALA with sPLA(2) was studied by fluorescent spectral analysis and circular dichroism studies. In vivo anti-inflammatory activity was checked using sPLA(2) induced mouse paw edema model. ALA but not DHLA inhibited purified sPLA(2) enzymes from V. russellii, N. naja and partially purified HAF, HPF and HS in a dose dependent manner. This data indicated that ALA is critical for inhibition. IC(50) value calculated for these enzymes ranges from 0.75 to 3.0 microM. The inhibition is independent of calcium and substrate concentration. Inflammatory sPLA(2) enzymes are more sensitive to inhibition by ALA than snake venom sPLA(2) enzymes. ALA quenched the fluorescence intensity of sPLA(2) enzyme in a dose dependent manner. Apparent shift in the far UV-CD spectra of sPLA(2) with ALA indicated change in its alpha-helical confirmation and these results suggest its direct interaction with the enzyme. ALA inhibits the sPLA(2) induced mouse paw edema in a dose dependent manner and confirms the sPLA(2) inhibitory activity in vivo also. These data suggest that ALA may act as an endogenous regulator of sPLA(2) enzyme activity and suppress inflammatory reactions. 相似文献
3.
Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA2 pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA2 sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA2s while metazoan representatives are similar with GIX PLA2 of the marine snail Conus magus. Members of GXIV and GIX PLA2s show modest overall sequence similarity (21–35%) but considerable motif conservation within the putative Ca2+-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA2s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA2 homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA2 deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA2s, two of which correspond to those localized in GXIV PLA2s. Phylogenetic analysis demonstrates that metazoan GIX PLA2s cluster separate from the bacterial and fungal GXIV PLA2s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA2 pfam06951. Duplicate PLA2 pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA2s and support the idea of the common evolutionary origin of GXIV and GIX PLA2 pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa. 相似文献
4.
The interfacial activation of porcine pancreatic phospholipase A(2) (PLA(2)) during the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes at different temperatures has been monitored by fluorescence changes of the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) lipid derivatives 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (C(12)-NBD-PC) and 12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)]dodecanoic acid (C(12)-NBD-FA) inserted in the substrate vesicles. These long-chain monitors, in contrast to the previously used C(6)-NBD-PC, detect latency times of PLA(2) action, similar to those measured by the classic titrimetric, pH-stat method. Interestingly, hydrolysis of the host vesicles results in a decrease in fluorescence not only of C(12)-NBD-PC, a substrate analog, but also of product derivative C(12)-NBD-FA. Ultrafiltration experiments show that C(12)-NBD-FA does not migrate to the aqueous phase upon hydrolysis of the host liposomes. Besides, in a simulated hydrolysis experiment in which increasing proportions of palmitic acid and 1-palmitoyl-sn-glycero-3-phosphocholine were cosonicated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C(12)-NBD-PC fluorescence was insensitive to products, whereas C(12)-NBD-FA did show a decreased emission intensity as in the actual hydrolysis experiments. The phenomenon is triggered above a critical concentration of products (10 mol%) suggesting that cosegregation of NBD-FA (either added as such or generated by hydrolysis of C(12)-NBD-PC) and products may be related to the decrease in fluorescence. Phase separation should create microdomains of increased C(12)-NBD-FA surface density and cause concentration quenching. In addition, and taking into account that the NBD group may be located near the interfacial region, it is possible that in segregating with products, the fluorescent moiety of C(12)-NBD-FA becomes exposed to microenvironments of higher surface polarity, which further decreases its quantum yield. 相似文献
5.
Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) is a group IIA phospholipase A2 which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA2 is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA2 mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA2 and the two mutants. Both native hnpsPLA2 and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA2 is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications. 相似文献
6.
The goal of the present study is to elucidate the effect of sphingomyelin on interfacial binding of Taiwan cobra phospholipase A2 (PLA2). Substitution of Asn-1 with Met caused a reduction in enzymatic activity and membrane-damaging activity of PLA2 toward phospholipid vesicles, while sphingomyelin exerted an inhibitory effect on the biological activities of native and mutated PLA2. Incorporation of sphingomyelin reduced membrane fluidity of phospholipid vesicles as evidenced by Laurdan fluorescence measurement. The results of self-quenching studies, binding of fluorescent probe, trinitrophenylation of Lys residues and fluorescence energy transfer between protein and lipid revealed that sphingomyelin altered differently membrane-bound mode of native and mutated PLA2. Moreover, it was found that PLA2 and N-terminally mutated PLA2 adopted different conformation and geometrical arrangement on binding with membrane bilayer. Nevertheless, the binding affinity of PLA2 and N-terminal mutant for phospholipid vesicles was not greatly affected by sphingomyelin. Together with the finding that mutation on N-terminus altered the gross conformation of PLA2, our data indicate that sphingomyelin modulates the mode of membrane binding of PLA2 at water/lipid interface, and suggest that the modulated effect of sphingomyelin depends on inherent structural elements of PLA2. 相似文献
7.
The secretory phospholipase A2-alpha from Arabidopsis thaliana (AtsPLA2-alpha), being one of the first plant sPLA2s obtained in purified state, has been characterised with respect to substrate preference and optimum conditions of catalysis. The optima of pH, temperature, and calcium concentration were similar to the parameters of secretory PLA2s from animals. However, substrate preferences markedly differed. In contrast to pancreatic PLA2s, AtsPLA2-alpha preferred zwitterionic phospholipids, and showed lower activity toward anionic phospholipids. In substrates with two identical fatty acid chains, AtsPLA2-alpha showed optimum activity toward phospholipids with decanoyl groups. In substrates with palmitoyl groups in sn-1 position, acyl chains with higher degree of unsaturation in sn-2 position were preferred, excluding arachidonic acid, showing the evolutionary adaptation of the enzyme to substrate composition in plants. Km values for short chain phospholipids were comparable to sPLA2s from animals, whereas k cat values were much smaller and interfacial activation was less important. 相似文献
8.
Secretory phospholipase A(2) (sPLA(2)) plays important roles in mediating various cellular processes, including cell proliferation, differentiation, apoptosis, and inflammatory response. In this study, we demonstrated that a basic sPLA(2) inhibits epidermal growth factor (EGF)-induced EGF receptor activation, as determined by autophosphorylation of EGF receptor, EGF-activated phospholipase D (PLD) activity, and phospholipase C-gamma(1) (PLC-gamma(1)) tyrosine phosphorylation in a human epidermoid carcinoma cell line, A-431. Treatment of cells with exogenous neutral sphingomyelinase (SMase) or a cell permeable ceramide analog, C(2)-ceramide, also caused similar inhibitory effects on EGF-induced activation of EGF receptor, tyrosine phosphorylation of PLC-gamma(1), and the activation of PLD. sPLA(2)-induced inhibition of EGF receptor was associated with arachidonic acid release, which was followed by an increase in intracellular ceramide formation. Both sPLA(2) and exogenous C(2)-ceramide are able to inhibit the proliferation of A-431. The data presented indicate for the first time that sPLA(2) downregulates the EGF receptor-mediated intracellular signal transduction that may be mediated by arachidonic acid and/or ceramide. 相似文献
9.
《朊病毒》2013,7(4):350-353
Precisely how the accumulation of PrPSc causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrPSc oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrPC, either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A2 (cPLA2). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA2 and synapse damage induced by cross-linked PrPC. We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA2 and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrPC, suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases. 相似文献
10.
Ramarao MK Shen MW Murphy EA Duan W Zhao Y McKew J Lee KL Thakker P Behnke ML Clark JD 《Analytical biochemistry》2008,383(2):217-225
Cytosolic phospholipase A2 alpha (cPLA2α, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA2α inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA2α and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA2α inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 °C and pH 7.4. The compounds tested bound cPLA2α with a 1:1 stoichiometry, and the dissociation constant Kd of the inhibitors calculated from the ITC experiments correlated well with the IC50 values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA2α was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA2α. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA2α was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics. 相似文献
11.
Persaud SJ Jones PM Roderigo-Milne HM Buchan AM Squires PE 《Biochemical and biophysical research communications》2003,300(4):889-893
Cytosolic phospholipase A(2)(cPLA(2)), an enzyme responsible for the generation of arachidonic acid, is located in the cytosolic compartment in most tissues and it translocates to membrane compartments when activated. We found that cPLA(2) distribution in pancreatic beta-cells is different from that of most other mammalian cells: it is evenly distributed throughout the beta-cell, in both cytoplasmic and nuclear compartments. Agents that increased intracellular Ca(2+) in the MIN6 beta-cell line also stimulated a redistribution of cPLA(2) immunoreactivity such that the majority of the enzyme moved from the nucleus to the cytoplasm. The time course of events was compatible with the elevation in Ca(2+) being responsible for translocation of cPLA(2). These observations suggest that cPLA(2) may be compartmentalised in unstimulated beta-cells, perhaps to limit its access to substrate prior to elevations in intracellular Ca(2+). 相似文献
12.
Richard C. Franson Lesley K. Harris Ramesh Raghupathi 《Molecular and cellular biochemistry》1989,88(1-2):155-159
Summary Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A2 activity (PLA2) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca2+-dependent PLA2 was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA, activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC50 for arachidonic acid was approx 65 M. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37°C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA2 inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA2s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury. 相似文献
13.
Lee SH Lee JG Kim JR Baek SH 《Biochemical and biophysical research communications》2007,364(4):996-1001
Although CpG containing DNA is an important regulator of innate immune responses via toll-like receptor 9 (TLR9), excessive activation of this receptor is detrimental to the host. Here, we show that cytosolic phospholipase A2 (cPLA2) activation is important for TLR9-mediated inducible nitric oxide synthase (iNOS) expression. Activation of TLR9 signaling by CpG induces iNOS expression and NO production. Inhibition of TLR9 blocked the iNOS expression and NO production. The CpG also stimulates cPLA2-hydrolyzed arachidonic acid (AA) release. Inhibition of cPLA2 activity by inhibitor attenuated the iNOS expression by CpG response. Additionally, knockdown of cPLA2 protein by miRNA also suppressed the CpG-induced iNOS expression. Furthermore, the CpG rapidly phosphorylates three MAPKs and Akt. A potent inhibitor for p38 MAPK or Akt blocked the CpG-induced AA release and iNOS expression. These results suggest that TLR9 activation stimulates cPLA2 activity via p38 or Akt pathways and mediates iNOS expression. 相似文献
14.
Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed. 相似文献
15.
Fluorescence measurements of the homologous proteins, notexin and PLA2 enzymes fromNaja naja atra, Naja nigricollis, and Hemachatus haemachatus venoms, showed that the wavelength of maximum emission and the quantum yield of their intrinsic fluorescence emission spectra were different. To verify the factors which affected their fluorescence characteristics, the dynamics of tryptophan residues in those homologous proteins were studied by quenching with acrylamide, iodide, and cesium. The degrees of exposure of tryptophanyl groups in notexin and PLA2 enzymes assessed by acrylamide quenching were found to be the major factor that determined their fluorescence characteristics. However, the positively charged groups surrounding tryptophan residues of PLA2 enzymes fromN. naja atra andN. nigricollis venoms might affect the quantum yield of their fluorophores. Tryptophan residues of notexin were in an environment with less fluctuation, which did not allow free diffusion of ionic quencher. This might render its typtophan residues to fluoresce at a shorter wavelength. These results suggested that the structural determinants affecting the intrinsic fluorescence emission of homologous proteins can be easily assessed by quenching studies. 相似文献
16.
María Elisa Mariani Marcos Ariel Villarreal Foo Cheung Ezequiel Pedro Marcos Leiva Ricardo Román Madoery Gerardo Daniel Fidelio 《Biochimie》2012
At the present, no secreted phospholipase A2 (sPLA2) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA2 from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca2+ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA2s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA2s including the novel enzymes from G. max. According to PLA2 superfamily, two of G. max sPLA2s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA2-XIA-1. We demonstrate that this mature sPLA2 of 114 residues had PLA2 activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA2 from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA2-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca+2 loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA2-XIA-1 structure. 相似文献
17.
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. 相似文献
18.
B. van den Berg M. Tessari R. Boelens R. Dijkman R. Kaptein G. H. de Haas H. M. Verheij 《Journal of biomolecular NMR》1995,5(2):110-121
Summary The three-dimensional structure of porcine pancreatic PLA2 (PLA2), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1–62 and 72–124 is 0.75±0.09 Å for the backbone atoms and 1.14±0.10 Å for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA2 and PLA2 in the ternary complex is 3.5 Å for the backbone atoms and 4.0 Å for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal -helix. Whereas free in solution Ala1, Leu2 and Trp3 are disordered, with the -amino group of Ala1 pointing out into the solvent, in the ternary complex these residues have an -helical conformation with the -amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA2. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA2 in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA2. 相似文献
19.
Chandra V Jasti J Kaur P Betzel Ch Srinivasan A Singh TP 《Journal of molecular biology》2002,320(2):215-222
This is the first structural evidence of alpha-tocopherol (alpha-TP) as a possible candidate against inflammation, as it inhibits phospholipase A2 specifically and effectively. The crystal structure of the complex formed between Vipera russelli phospholipase A2 and alpha-tocopherol has been determined and refined to a resolution of 1.8 A. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one alpha-tocopherol molecule, which is bound specifically to one of them. The phospholipase A2 molecules interact extensively with each other in the crystalline state. The two molecules were found in a stable association in the solution state as well, thus indicating their inherent tendency to remain together as a structural unit, leading to significant functional implications. In the crystal structure, the most important difference between the conformations of two molecules as a result of their association pertains to the orientation of Trp31. It may be noted that Trp31 is located at the mouth of the hydrophobic channel that forms the binding domain of the enzyme. The values of torsion angles (phi, psi, chi(1) and chi(2)) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94 degrees, -30 degrees, -66 degrees, 116 degrees and -128 degrees, 170 degrees, -63 degrees, -81 degrees, respectively. The conformation of Trp31 in molecule A is suitable for binding, while that in B hinders the passage of the ligand to the binding site. Consequently, alpha-tocopherol is able to bind to molecule A only, while the binding site of molecule B contains three water molecules. In the complex, the aromatic moiety of alpha-tocopherol is placed in the large space at the active site of the enzyme, while the long hydrophobic channel in the enzyme is filled by hydrocarbon chain of alpha-tocopherol. The critical interactions between the enzyme and alpha-tocopherol are generated between the hydroxyl group of the six-membered ring of alpha-tocopherol and His48 N(delta1) and Asp49 O(delta1) as characteristic hydrogen bonds. The remaining part of alpha-tocopherol interacts extensively with the residues of the hydrophobic channel of the enzyme, giving rise to a number of hydrophobic interactions, resulting in the formation of a stable complex. 相似文献
20.
Touqui L Paya M Thouron F Guiyoule A Zarantonelli ML Leduc D Wu Y Taha MK Alonso JM 《FEBS letters》2005,579(22):4923-4927
Induction of type-IIA secreted phospholipase A2 (sPLA2-IIA) expression by bacterial components other than lipopolysaccharide has not been previously investigated. Here, we show that exposure of alveolar macrophages (AM) to Neisseria meningitidis or its lipooligosaccharide (LOS) induced sPLA2-IIA synthesis. However, N. meningitidis mutant devoid of LOS did not abolish this effect. In addition, a pili-defective mutant exhibited significantly lower capacity to stimulate sPLA2-IIA synthesis than the wild-type strain. Moreover, pili isolated from a LOS-defective strain induced sPLA2-IIA expression and nuclear factor kappa B (NF-kappaB) activation. These data suggest that pili are potent inducers of sPLA2-IIA expression by AM, through a NF-kappaB-dependent process. 相似文献