Structure, function, and regulation of group V phospholipase A(2)

The hydrolysis of membrane phospholipid by phospholipase A(2) (PLA(2)) is a key step in the production of inflammatory eicosanoids. Recent cell studies have shown that secretory group V PLA(2) (gVPLA(2)) is involved in agonist-induced eicosanoid biosynthesis in mouse P388D1 cell line, mast cells, and transfected HEK 293 cells. gVPLA(2) is homologous to other group II PLA(2) family members but has distinctive enzymatic properties, including its activity to effectively hydrolyze phosphatidylcholine (PC) vesicles and the outer plasma membrane of mammalian cells. Mutational studies showed that gVPLA(2) has a unique structure that allows effective binding to PC membranes and efficient catalysis of an active-site-bound PC substrate. Thanks to this unique structure and activity, exogenously added gVPLA(2) can induce the eicosanoid biosynthesis in unstimulated inflammatory cells, including human neutrophils and eosinophils, suggesting that it might be able to trigger inflammatory responses under certain physiological conditions. Extensive structure-function and cell studies showed that gVPLA(2) could act directly on the outer plasma membranes of neutrophils and eosinophils. The release of fatty acids and lysophospholipids from the cell surfaces induces the translocation and activation of cytosolic PLA(2) and 5-lipoxygenase, resulting in the leukotriene synthesis. In case of neutrophils, induction of leukotriene B(4) synthesis by gVPLA(2) leads to the phosphorylation of cytosolic PLA(2) by a leukotriene B(4) receptor and MAP kinase-mediated mechanism. Finally, heparan sulfate proteoglycans in neutrophils appear to play a role of internalizing and degrading the cell surface-bound gVPLA(2) to protect the cells from extensive lipolytic damage.     

Structural analysis of secretory phospholipase A2 from Clonorchis sinensis: therapeutic implications for hepatic fibrosis

Hepatic fibrosis is a common complication of the infection by the parasite, Clonorchis sinensis. There is a high incidence of this disease in the Asian countries with an increased risk of conversion to cancer. A secretory phospholipase A(2) (PLA(2)) enzyme from the parasite is implicated in the pathology. This is an attractive drug target in the light of extensive structural characterization of this class of enzyme. In this study, the structure of the enzyme was modeled based on its sequence homology to the group III bee venom PLA(2). On analysis, the overall structure essentially is comprised of three helices, two sets of β-wings and an elongated C-terminal extension. The structure is stabilized by four disulfide bonds. The structure is comprised of a calcium binding loop, active site and a substrate binding hydrophobic channel. The active site of the enzyme shows the classical features of PLA(2) with the participation of the three residues: histidine-aspartic acid-tyrosine in hydrogen bond formation. This is an interesting variation from the house keeping group III PLA(2) enzyme of human which has a histidine-aspartic acid and phenylalanine arrangement at the active site. This difference is therefore an important structural parameter that can be exploited to design specific inhibitor molecules against the pathogen PLA(2). Likewise, there are certain unique structural features in the hydrophobic channel and the putative membrane binding surface of the PLA(2) from Clonorchis sinensis that not only help understand the mechanism of action but also provide knowledge for a targeted therapy of liver fibrosis caused by the parasite.     

Structural effects of covalent inhibition of phospholipase A2 suggest allosteric coupling between membrane binding and catalytic sites

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 membrane-binding 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 alpha-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 membrane-binding 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 alpha-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.     

An enzymatically active chimeric protein containing the hydrophilic form of NADPH-cytochrome P450 reductase fused to the membrane-binding domain of cytochrome b5.

The microsomal flavoprotein NADPH-cytochrome P450 reductase (CPR) contains an N-terminal hydrophobic membrane-binding domain required for reconstitution of hydroxylation activities with cytochrome P450s. In contrast, cytochrome b5 (b5) contains a C-terminal hydrophobic membrane-binding domain required for interaction with P450s. We have constructed, expressed and purified a chimeric flavoprotein (hdb5-CPR) where the C-terminal 45 amino acid residues of b5 have replaced the N-terminal 56 amino acid domain of CPR. This hybrid flavoprotein retains the catalytic properties of the native CPR and is able to reconstitute fatty acid and steroid hydroxylation activities with CYP4A1 and CYP17A. However hdb5-CPR is much less effective than CPR for reconstituting activity with CYP3A4. We conclude that differences on the surface of the P450s reflect unique and specific information essential for the recognition needed to establish reactions of intermolecular electron transfer from the flavoprotein CPR.     

Three-dimensional structure of a presynaptic neurotoxic phospholipase A2 from Daboia russelli pulchella at 2.4 A resolution

The phospholipase A(2 )from Daboia russelli pulchella (DPLA(2)) is the only known member of subclass II of group IIA. The three-dimensional structure of this presynaptic neurotoxic DPLA(2) enzyme has been determined at 2.4 A resolution. The structure was determined by the molecular replacement method using the model Crotalus atrox, and refined using X-PLOR to a final R-factor of 18.8 % for all data in the resolution range 20.0 A-2.4 A. The final refined model comprises 1888 atoms from two crystallographically independent protein molecules and 160 water oxygen atoms. The overall folding of DPLA(2), with three long helices and two short antiparallel beta-strands is grossly similar to those observed for other PLA(2)s. In the present structure, the calcium binding site is empty but the conformation of the calcium binding loop is similar to those observed in the calcium bound states. Two spatially adjacent regions of residues 55-61 (a typical beta-turn I) and 83-94 (a well defined loop) are remarkably different in conformation, electrostatic characteristics and inter-segmental interactions from those found in non-neurotoxic PLA(2)s. Yet another striking structural feature in DPLA(2 )pertains to the stretch of residues 53-77, which has a series of positively charged residues protruding outwardly. The above segment is presumed to be involved in the anticoagulant activity. A unique hydrophobic patch including residues Leu17, Ala18, Ile19, Pro20, Phe106 and Leu110 is found on the surface together with an equally emphatic region of -OH groups containing residues such as Ser21, Tyr22, Ser23, Ser24, Tyr25 and Tyr28. The interactions between two molecules of DPLA(2) in the asymmetric unit are remarkably different from those observed in the standard dimers and trimers of PLA(2)s, leaving the enzyme's active site fully exposed for enzyme-substrate reactions, it makes this structure one of the most favourable examples for structure-based drug design through soaking experiments.     

Determination of the Substrate Specificity of Protein-tyrosine Phosphatase TULA-2 and Identification of Syk as a TULA-2 Substrate

TULA-1 (UBASH3A/STS-2) and TULA-2 (p70/STS-1) represent a novel class of protein-tyrosine phosphatases. Previous studies suggest that TULA-2 is sequence-selective toward phosphotyrosyl (Tyr(P)) peptides. In this work the substrate specificity of TULA-1 and -2 was systematically evaluated by screening a combinatorial Tyr(P) peptide library. Although TULA-1 showed no detectable activity toward any of the Tyr(P) peptides in the library, TULA-2 recognizes two distinct classes of Tyr(P) substrates. On the N-terminal side of Tyr(P), the class I substrates contain a proline at the Tyr(P)−1 position, a hydrophilic residue at the Tyr(P)−2 position, and aromatic hydrophobic residues at positions Tyr(P)−3 and beyond. The class II substrates typically contain two or more acidic residues, especially at Tyr(P)−1 to Tyr(P)−3 positions, and aromatic hydrophobic residues at other positions. At the C-terminal side of Tyr(P), TULA-2 generally prefers acidic and aromatic residues. The library screening results were confirmed by kinetic analysis of representative peptides selected from the library as well as Tyr(P) peptides derived from various Tyr(P) proteins. TULA-2 is highly active toward peptides corresponding to the Tyr(P)-323 and Tyr(P)-352 sites of Syk, and the Tyr(P)-397 site of focal adhesion kinase and has lower activity toward other Tyr(P) sites in these proteins. In glycoprotein VI-stimulated platelets, knock-out of the TULA-2 gene significantly increased the phosphorylation level of Syk at Tyr-323 and Tyr-352 sites and to a lesser degree at the Tyr-525/526 sites. These results suggest that Syk is a bona fide TULA-2 substrate in platelets.     

Single replacement constructs of all hydroxyl, basic, and acidic amino acids identify new function and structure-sensitive regions of the mitochondrial phosphate transport protein

The phosphate transport protein (PTP) catalyzes the proton cotransport of phosphate into the mitochondrial matrix. It functions as a homodimer, and thus residues of the phosphate and proton pores are somewhat scattered throughout the primary sequence. With 71 new single mutation per subunit PTPs, all its hydroxyl, basic, and acidic residues have now been replaced to identify these essential residues. We assayed the initial rate of pH gradient-dependent unidirectional phosphate transport activity and the liposome incorporation efficiency (LIE) of these mutants. Single mutations of Thr79, Tyr83, Lys90, Tyr94, and Lys98 inactivate transport. The spacings between these residues imply that they are located along the same face of transmembrane (TM) helix B, requiring an extension of its current model C-terminal domain by 10 residues. This extension superimposes very well onto the shorter bovine PTP helix B, leaving a 15-residue hydrophobic extension of the yeast helix B N-terminus. This is similar to the helix D and F regions of the yeast PTP. Only one transport-inhibiting mutation is located within loops: Ser158Thr in the matrix loop between helices C and D. All other transport-inhibiting mutations are located within the TM helices. Mutations that yield LIEs of <6% are all, except for four, within helices. The four exceptions are Tyr12Ala near the PTP N-terminus and Arg159Ala, Glu163Gln, and Glu164Gln in the loop between helices C and D. The PTP C-terminal segment beyond Thr214 at the N-terminus of helix E has 11 mutations with LIEs >20% and none with LIE <6%. Mutations with LIEs >20% are located near the ends of all the TM helices except TM helix D. Only a few mutations alter PTP structure (LIE) and also affect PTP transport activity. A novel observation is that Ser4Ala blocks the formation of PTP bacterial inclusion bodies.     

Phospholipase A2 as a target protein for nonsteroidal anti-inflammatory drugs (NSAIDS): crystal structure of the complex formed between phospholipase A2 and oxyphenbutazone at 1.6 A resolution

Phospholipase A(2) (PLA(2); EC 3.1.1.4) is a key enzyme involved in the production of proinflammatory mediators known as eicosanoids. The binding of the substrate to PLA(2) occurs through a well-formed hydrophobic channel. To determine the viability of PLA(2) as a target molecule for the structure-based drug design against inflammation, arthritis, and rheumatism, the crystal structure of the complex of PLA(2) with a known anti-inflammatory compound oxyphenbutazone (OPB), which has been determined at 1.6 A resolution. The structure has been refined to an R factor of 0.209. The structure contains 1 molecule each of PLA(2) and OPB with 2 sulfate ions and 111 water molecules. The binding studies using surface plasmon resonance show that OPB binds to PLA(2) with a dissociation constant of 6.4 x 10(-8) M. The structure determination has revealed the presence of an OPB molecule at the binding site of PLA(2). It fits well in the binding region, thus displaying a high level of complementarity. The structure also indicates that OPB works as a competitive inhibitor. A large number of hydrophobic interactions between the enzyme and the OPB molecule have been observed. The hydrophobic interactions involving residues Tyr(52) and Lys(69) with OPB are particularly noteworthy. Other residues of the hydrophobic channel such as Leu(3), Phe(5), Met(8), Ile(9), and Ala(18) are also interacting extensively with the inhibitor. The crystal structure clearly reveals that the binding of OPB to PLA(2) is specific in nature and possibly suggests that the basis of its anti-inflammatory effects may be due to its binding to PLA(2) as well.     

Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 A resolution

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and partial characterization of coxsackievirus B3 2A protease expressed in Escherichia coli

Reported here is the overexpression, purification and partial characterization of recombinant coxsakievirus B3 2A protease (CVB3 2A(pro)) from bacterial cells transformed with a plasmid containing the CVB3 2A(pro) cDNA sequences. The structural investigation showed that the protein contains mostly beta-strand elements and requires Zn(2+) ions as a structural component which appeared to be inhibitory if added exogenously. The purified enzyme activity was optimal at 4 degrees C and had a short half-life at physiological temperature. This feature can be the result of the presence of a high content of beta-structure and also hydrophobic residues in its structure.     

Cellular arachidonate-releasing function and inflammation-associated expression of group IIF secretory phospholipase A2

Here we report the cellular arachidonate (AA)-releasing function of group IIF secretory phospholipase A(2) (sPLA(2)-IIF), a sPLA(2) enzyme uniquely containing a longer C-terminal extension. sPLA(2)-IIF increased spontaneous and stimulus-dependent release of AA, which was supplied to downstream cyclooxygenases and 5-lipoxygenase for eicosanoid production. sPLA(2)-IIF also enhanced interleukin 1-stimulated expression of cyclooxygenase-2 and microsomal prostaglandin E synthase. AA release by sPLA(2)-IIF was facilitated by oxidative modification of cellular membranes. Cellular actions of sPLA(2)-IIF occurred independently of the heparan sulfate proteoglycan glypican, which acts as a functional adaptor for other group II subfamily sPLA(2)s. Confocal microscopy revealed the location of sPLA(2)-IIF on the plasma membrane. The unique C-terminal extension was crucial for its plasma membrane localization and optimal cellular functions. sPLA(2)-IIF expression was increased in various tissues from lipopolysaccharide-treated mice and in ears of mice with experimental atopic dermatitis. In human rheumatoid arthritic joints, sPLA(2)-IIF was detected in synovial lining cells, capillary endothelial cells, and plasma cells. These results suggest that sPLA(2)-IIF is a potent regulator of AA metabolism and participates in the inflammatory process under certain conditions.     

Mutational analysis of the role of tryptophan residues in an antimicrobial peptide

Antimicrobial peptides belonging to the pediocin-like family of bacteriocins (class IIa bacteriocins) produced by lactic acid bacteria contain several tryptophan residues that are highly conserved. Since tryptophan residues in membrane proteins are often positioned in the membrane-water interface, we hypothesized that Trp residues in bacteriocins could be important determinants of the structure of membrane-bound peptides and of anti-microbial activity. To test this hypothesis, the effects of mutating each of the 3 tryptophan residues (Trp18, Trp33, and Trp41) in the 43-residue pediocin-like bacteriocin sakacin P were studied. Trp18 and Trp33 are located at each end of an amphihilic alpha-helix, whereas Trp41 is near the end of an unstructured C-terminal tail. Replacement of Trp33 with the hydrophobic residues Leu and Phe had marginal effects on activity, whereas replacement with the more polar Tyr and Arg reduced activity 10-20 and 500-1000 times, respectively, indicating that Trp33 and the C-terminal part of the helix interact with the hydrophobic core of the membrane. Any mutation of Trp18 and Trp41 reduced activity, indicating that these two residues play unique roles. Substitutions with other aromatic residues were the least deleterious, indicating that both Trp18 and Trp41 interact with the membrane-water interface. The suggested locations of the three Trp residues are compatible with a structural model in which the helix and the C-terminal tail form a hairpin-like structure, bringing Trp18 and Trp41 close to each other in the interface, and placing Trp33 in the hydrophobic core of the membrane. Indeed, the deleterious effect of the W18L and W41L mutations could be overcome by stabilizing the hairpin-like structure by introduction of a disulfide bridge between residues 24 and 44. These results provide a basis for a refined structural model of pediocin-like bacteriocins and highlight the unique role that tryptophan residues can play in membrane-interacting peptides.     

Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A

Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.     

Role of S'1 loop residues in the substrate specificities of pepsin A and chymosin

Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.     

Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path

GA (glucoamylase) hydrolyses starch and polysaccharides to beta-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with beta-cyclodextrin (SBD-betaCD) and maltoheptaose (SBD-G7) were determined. Two carbohydrate binding sites, I (Trp(47)) and II (Tyr(32)), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr(32) was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.     

Plasma membrane and nuclear localization of G protein coupled receptor kinase 6A

G protein-coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.     

LDL phospholipid hydrolysis produces modified electronegative particles with an unfolded apoB-100 protein

Electronegative low density lipoprotein (LDL(-)) formation that structurally resembles LDL(-) isolated from plasma was evaluated after LDL treatment with snake venom phospholipase A(2) (PLA(2)). PLA(2) treatment of LDL increased its electrophoretic mobility in proportion to the amount of LDL(-) formed without evidence of lipid peroxidation. These changes dose-dependently correlated with the degree of phospholipid hydrolysis. Strong immunoreactivity of LDL(-) subfraction from plasma and PLA(2)-treated LDL (PLA(2)-LDL) to amyloid oligomer-specific antibody was observed. Higher beta-strand structural content and unfolding proportionate to the loss of alpha-helical structure of apolipoprotein B-100 (apoB-100) of LDL(-) isolated from both native and PLA(2)-LDLs was demonstrated by circular dichroism (CD) spectropolarimetry. These structural changes resembled the characteristics of some oxidatively modified LDLs and soluble oligomeric aggregates of amyloidogenic proteins. PLA(2)-LDL was also more susceptible to nitration by peroxynitrite, likely because of exposure of otherwise inaccessible hydrophilic and hydrophobic domains arising from apoB-100 unfolding. This was also demonstrated for plasma LDL(-). In contrast, PLA(2)-LDL was more resistant to copper-mediated oxidation that was reversed upon the addition of small amounts of unsaturated fatty acids. The observed similarities between PLA(2)-LDL(-)-derived LDL(-) and plasma LDL(-) implicate a role for secretory PLA(2) in producing modified LDL(-) that is facilitated by unfolding of apoB-100.     

The interaction of phospholipase A2 with a phospholipid bilayer: coarse-grained molecular dynamics simulations

A number of membrane-active enzymes act in a complex environment formed by the interface between a lipid bilayer and bulk water. Although x-ray diffraction studies yield structures of isolated enzyme molecules, a detailed characterization of their interactions with the interface requires a measure of how deeply such a membrane-associated protein penetrates into a lipid bilayer. Here, we apply coarse-grained (CG) molecular dynamics (MD) simulations to probe the interaction of porcine pancreatic phospholipase A2 (PLA2) with a lipid bilayer containing palmitoyl-oleoyl-phosphatidyl choline and palmitoyl-oleoyl-phosphatidyl glycerol molecules. We also used a configuration from a CG-MD trajectory to initiate two atomistic (AT) MD simulations. The results of the CG and AT simulations are evaluated by comparison with available experimental data. The membrane-binding surface of PLA2 consists of a patch of hydrophobic residues surrounded by polar and basic residues. We show this proposed footprint interacts preferentially with the anionic headgroups of the palmitoyl-oleoyl-phosphatidyl glycerol molecules. Thus, both electrostatic and hydrophobic interactions determine the location of PLA2 relative to the bilayer. From a general perspective, this study demonstrates that CG-MD simulations may be used to reveal the orientation and location of a membrane-surface-bound protein relative to a lipid bilayer, which may subsequently be refined by AT-MD simulations to probe more detailed interactions.     

Inducing rigid local structure around the zinc-binding region by hydrophobic interactions enhances the homotrimerization and apoptotic activity of zinc-free TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), existing as homotrimer in solution, contains a unique zinc-binding site coordinated by three Cys230 residues at the tip of trimeric interface. TRAIL mutant with replacements of Cys230 with Ala (TRAIL(C230A)) negligibly formed trimeric structure and showed no apoptotic activity. Here, to elucidate the relationship between the trimeric stability and the apoptotic activity of TRAIL(C230A), we rationally designed mutations to induce homotrimerization of TRAIL(C230A) by substituting for the three residues involved in hydrogen bonding (Tyr183 and Tyr243) and putative repulsive electrostatic (Arg227) interactions at the buried trimeric interface into hydrophobic residues, like Y183F, Y243F, and R227I. The TRAIL(C230A)-derived mutants exhibited enhanced homotrimerization, but only the mutants containing R227I exhibited significant apoptosis-inducing activity in cancer cells. These results, together with the induction of rigid local structure around the zinc-binding region by R227I in TRAIL(C230A), suggest that ordered, rigid structure around the zinc-binding region is critical for the homotrimerization and apoptotic activity of TRAIL.