Design of specific peptide inhibitors for group I phospholipase A2: structure of a complex formed between phospholipase A2 from Naja naja sagittifera (group I) and a designed peptide inhibitor Val-Ala-Phe-Arg-Ser (VAFRS) at 1.9 A resolution reveals unique features

Phospholipase A(2) (PLA(2)) (E. C. 3.1.1.4) is a common enzyme in the two-way cascade mechanism leading to the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to the membrane surface and hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before its cleavage. To regulate the production of proinflammatory compounds, a specific peptide inhibitor Val-Ala-Phe-Arg-Ser (VAFRS) for the group I PLA(2) enzymes has been designed and synthesized. PLA(2) was isolated from Indian cobra (Naja naja sagittifera) venom and purified to homogeneity. The binding studies indicated the K(i) value of 1.02 +/- 0.10 x 10(-8) M. The purified PLA(2) samples and the designed inhibitor VAFRS were cocrystallized. The crystal structure of the complex was determined and refined to 1.9 A resolution. The peptide binds to PLA(2) at the active site and fills the hydrophobic channel completely. However, its placement with respect to the channel is in the opposite direction as compared to those observed in group II PLA(2)'s. Furthermore, the predominant intermolecular interactions involve strong electrostatic interactions between the side chains of peptide Arg and Asp 49 of PLA(2) together with a number of van der Waals interactions with other residues. A good number of observed interactions between the peptide and the protein indicate the significance of a structure-based drug design approach. The novel factor in the present sequence of the peptide is related to the introduction of a positively charged residue at the C-terminal part of the peptide.     

Crystal structures of the complexes of a group IIA phospholipase A2 with two natural anti-inflammatory agents, anisic acid, and atropine reveal a similar mode of binding

Secretory low molecular weight phospholipase A(2)s (PLA(2)s) are believed to be involved in the release of arachidonic acid, a precursor for the biosynthesis of pro-inflammatory eicosanoids. Therefore, the specific inhibitors of these enzymes may act as potent anti-inflammatory agents. Similarly, the compounds with known anti-inflammatory properties should act as specific inhibitors. Two plant compounds, (a) anisic acid (4-methoxy benzoic acid) and (b) atropine (8-methyl-8-azabicyclo oct-3-hydroxy-2-phenylpropanoate), have been used in various inflammatory disorders. Both compounds (a) and (b) have been found to inhibit PLA(2) activity having binding constants of 4.5 x 10(-5) M and 2.1 x 10(-8) M, respectively. A group IIA PLA(2) was isolated and purified from the venom of Daboia russelli pulchella (DRP) and its complexes were made with anisic acid and atropine. The crystal structures of the two complexes (i) and (ii) of PLA(2) with compounds (a) and (b) have been determined at 1.3 and 1.2 A resolutions, respectively. The high-quality observed electron densities for the two compounds allowed the accurate determinations of their atomic positions. The structures revealed that these compounds bound to the enzyme at the substrate - binding cleft and their positions were stabilized by networks of hydrogen bonds and hydrophobic interactions. The most characteristic interactions involving Asp 49 and His 48 were clearly observed in both complexes, although the residues that formed hydrophobic interactions with these compounds were not identical because their positions did not exactly superimpose in the large substrate-binding hydrophobic channel. Owing to a relatively small size, the structure of anisic acid did not alter upon binding to PLA(2), while that of atropine changed significantly when compared with its native crystal structure. The conformation of the protein also did not show notable changes upon the bindings of these ligands. The mode of binding of anisic acid to the present group II PLA(2) is almost identical to its binding with bovine pancreatic PLA(2) of group I. On the other hand, the binding of atropine to PLA(2) is similar to that of another plant alkaloid aristolochic acid.     

Active compound from the leaves of Vitex negundo L. shows anti-inflammatory activity with evidence of inhibition for secretory Phospholipase A(2) through molecular docking

Novel compounds with significant medicinal properties have gained much interest in therapeutic approaches for treating various inflammatory disorders like arthritis, odema and snake bites and the post-envenom (impregnating with venom) consequences. Inflammation is caused by the increased concentration of secretory Phospholipases A(2) (sPLA(2)s) at the site of envenom. A novel compound Tris(2,4-di-tert-butylphenyl) phosphate (TDTBPP) was isolated from the leaves of Vitex negundo and the crystal structure was reported recently. The acute anti-inflammatory activity of TDTBPP was assessed by Carrageenan-induced rat paw odema method. TDTBPP reduced the raw paw odema volume significantly at the tested doses of 50 mg/kg and 70 mg/kg body weight. Molecular docking studies were carried out with the X-ray crystal structures of Daboia russelli pulchella's (Vipera russelli, Indian Russell's viper) venom sPLA(2) and Human non-pancreatic secretory PLA(2) (Hnps PLA(2)) as targets to illustrate the antiinflammatory and antidote activities of TDTBPP. Docking results showed hydrogen bond (H-bond) interaction with Lys69 residue lying in the anti-coagulant loop of D. russelli's venom PLA(2), which is essential in the catalytic activity of the enzyme and hydrophobic interactions with the residues at the binding site (His48, Asp49). Docking of TDTBPP with Hnps PLA(2) structure showed coordination with calcium ion directly as well as through the catalytically important water molecule (HOH1260) located at the binding site.     

Interaction mode of n-dodecylphosphorylcholine, a substrate analogue, with bovine pancreas phospholipase A2 as determined by X-ray crystal analysis.

Three-dimensional structure of a bovine pancreas phospholipase A2 (PLA2) crystal complexed with n-dodecylphosphorylcholine (n-C12PC), a substrate-type inhibitor, has been determined by the X-ray diffraction method. The present conventional R value is 0.275 at 2.3A resolution. The binding mode of n-C12PC to the PLA2 was clearly indicated, where the dodecyl chain was stably held by the hydrophobic contacts with the N-terminal region of PLA2 (Leu-2, Phe-5, and Ile-9), and the choline moiety was contacted with the hydrophobic space created by the side chains of Lys-53 and 56. The present result indicates that remarkable changes from the native PLA2 structure are caused at the N-terminal and middle (residues 60 to 70) regions by the binding of n-C12PC to the enzyme.     

Structural basis of phospholipase A2 inhibition for the synthesis of prostaglandins by the plant alkaloid aristolochic acid from a 1.7 A crystal structure

This is the first structural observation of a plant product showing high affinity for phospholipase A(2) and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A(2) and a plant alkaloid aristolochic acid has been determined and refined to 1.7 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, beta-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp(31). Trp(31) is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp(31) is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp(31) in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His(48) and Asp(49).     

Asp49 phospholipase A(2)-elaidoylamide complex: a new mode of inhibition

The inhibition of phospholipase A(2)s (PLA(2)s) is of pharmacological and therapeutic interest because these enzymes are involved in several inflammatory diseases. Elaidoylamide is a powerful inhibitor of a neurotoxic PLA(2) from the Vipera ammodytes meridionalis venom. The X-ray structure of the enzyme-inhibitor complex reveals a new mode of Asp49 PLA(2) inhibition by a fatty acid hydrocarbon chain. The structure contains two identical homodimers in the asymmetric unit. In each dimer one subunit is rotated by 180 degrees with respect to the other and the two molecules are oriented head-to-tail. One molecule of elaidoylamide is bound simultaneously to the substrate binding sites of two associated neurotoxic phospholipase A(2) molecules. The inhibitor binds symmetrically to the hydrophobic channels of the two monomers. The structure can be used to design anti-inflammatory drugs.     

First structural evidence of a specific inhibition of phospholipase A2 by alpha-tocopherol (vitamin E) and its implications in inflammation: crystal structure of the complex formed between phospholipase A2 and alpha-tocopherol at 1.8 A resolution

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.     

Differential roles of ionic, aliphatic, and aromatic residues in membrane-protein interactions: a surface plasmon resonance study on phospholipases A2

The roles of cationic, aliphatic, and aromatic residues in the membrane association and dissociation of five phospholipases A(2) (PLA(2)), including Asp-49 PLA(2) from the venom of Agkistrodon piscivorus piscivorus, acidic PLA(2) from the venom of Naja naja atra, human group IIa and V PLA(2)s, and the C2 domain of cytosolic PLA(2), were determined by surface plasmon resonance analysis. Cationic interfacial binding residues of A. p. piscivorus PLA(2) (Lys-10) and human group IIa PLA(2) (Arg-7, Lys-10, and Lys-16), which mediate electrostatic interactions with anionic membranes, primarily accelerate the membrane association. In contrast, an aliphatic side chain of the C2 domain of cytosolic PLA(2) (Val-97), which penetrates into the hydrophobic core of the membrane and forms hydrophobic interactions, mainly slows the dissociation of membrane-bound protein. Aromatic residues of human group V PLA(2) (Trp-31) and N. n. atra PLA(2) (Trp-61, Phe-64, and Tyr-110) contribute to both membrane association and dissociation steps, and the relative contribution to these processes depends on the chemical nature and the orientation of the side chains as well as their location on the interfacial binding surface. On the basis of these results, a general model is proposed for the interfacial binding of peripheral proteins, in which electrostatic interactions by ionic and aromatic residues initially bring the protein to the membrane surface and the subsequent membrane penetration and hydrophobic interactions by aliphatic and aromatic residues stabilize the membrane-protein complexes, thereby elongating the membrane residence time of protein.     

Comparative analysis of the sequences and structures of HIV-1 and HIV-2 proteases

The different isolates available for HIV-1 and HIV-2 were compared for the region of the protease (PR) sequence, and the variations in amino acids were analyzed with respect to the crystal structure of HIV-1 PR with inhibitor. Based on the extensive homology (39 identical out of 99 residues), models were built of the HIV-2 PR complexed with two different aspartic protease inhibitors, acetylpepstatin and a renin inhibitor, H-261. Comparison of the HIV-1 PR crystal structure and the HIV-2 PR model structure and the analysis of the changes found in different isolates showed that correlated substitutions occur in the hydrophobic interior of the molecule and at surface residues involved in ionic or hydrogen bond interactions. The substrate binding residues of HIV-1 and HIV-2 PRs show conservative substitutions of four residues. The difference in affinity of HIV-1 and HIV-2 PRs for the two inhibitors appears to be due in part to the change of Val 32 in HIV-1 PR to Ile in HIV-2 PR.     

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.     

Crystal structure of the complex formed between a group I phospholipase A2 and a naturally occurring fatty acid at 2.7 A resolution

This is the first evidence of a naturally bound fatty acid to a group I Phospholipase A(2) (PLA(2)) and also to a PLA(2) with Asp 49. The fatty acid identified as n-tridecanoic acid is observed at the substrate recognition site of PLA(2) hydrophobic channel. The complex was isolated from the venom of Bungarus caeruleus (Common Indian Krait). The primary sequence of the PLA(2) was determined using the cDNA method. Three-dimensional structure has been solved by the molecular replacement method and refined using the CNS package to a final R factor of 19.8% for the data in the resolution range from 20.0 to 2.7 A. The final refined model is comprised of 912 protein atoms, one sodium ion, one molecule of n-tridecanoic acid, and 60 water molecules. The sodium ion is located in the calcium-binding loop with a sevenfold coordination. A characteristic extra electron density was observed in the hydrophobic channel of the enzyme, into which a molecule of n-tridecanoic acid was clearly fitted. The MALDI-TOF measurements of the crystals had earlier indicated an increase in the molecular mass of PLA(2) by 212 Da over the native PLA(2). A major part of the ligand fits well in the binding pocket and interacts directly with His 48 and Asp 49. Although the overall structure of PLA(2) in the present complex is similar to the native structure reported earlier, it differs significantly in the folding of its calcium-binding loop.     

Exploring the structure characteristics and major channels of cytochrome P450 2A6, 2A13, and 2E1 with pilocarpine

The majority of cytochromes P450 play a critical role in metabolism of endogenous and exogenous substrates, some of its products are carcinogens. Therefore, inhibition of P450 enzymes activity can promote the detoxification and elimination of chemical carcinogens. In this study, molecular dynamics (MD) simulations and adaptive steered molecular dynamics (ASMD) simulations were performed to explore the structure features and channel dynamics of three P450 isoforms 2A6, 2A13, and 2E1 bound with the common inhibitor pilocarpine. The binding free energy results combined with the PMF calculations give a reasonable ranking of binding affinity, which are consistent with the experimental data. Our results uncover how a sequence divergence of different CYP2 enzymes causes individual variations in major channel selections. On the basis of channel bottleneck and energy decomposition analysis, we propose a gating mechanism of their respective major channels in three enzymes, which may be attributed to a reversal of Phe209 in CYP2A6/2A13, as well as the rotation of Phe116 and Phe298 in CYP2E1. The hydrophobic residues not only make strong hydrophobic interactions with inhibitor, but also act as gatekeeper to regulate the opening of channel. The present study provides important insights into the structure–function relationships of three cytochrome P450s and the molecular basis for development of potent inhibitors.     

The three-dimensional structure of notexin, a presynaptic neurotoxic phospholipase A2 at 2.0 A resolution.

The three-dimensional structure of notexin has been solved by molecular replacement methods. The structure has been refined at 2.0 A resolution to a crystallographic R-value of 16.5% with good stereo-chemistry. The core of the protein is very similar to other phospholipase A2s (PLA2 s) but several parts of the molecule are distinctly different. The most significant differences from PLA2 s from bovine pancreas and rattlesnake occur in the stretches 56-80 and 85-89. Residue 69, which has been shown to be important for phospholipase binding, has a different conformation and different interactions than in other known PLA2s. The C alpha positions for residues 86-88 differ by about 6 A from both the bovine and the rattlesnake enzyme. The crystals contain no Ca2+ ions. Instead, a water molecule occupies the calcium site.     

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.     

Identification of a novel binding site for calmodulin in ammodytoxin A, a neurotoxic group IIA phospholipase A2.

The molecular mechanism of the presynaptic neurotoxicity of snake venom phospholipases A2 (PLA2s) is not yet fully elucidated. Recently, new high-affinity binding proteins for PLA2 toxins have been discovered, including the important intracellular Ca2+ sensor, calmodulin (CaM). In the present study, the mode of interaction of group IIA PLA2s with the Ca2+-bound form of CaM was investigated by mutational analysis of ammodytoxin A (AtxA) from the long-nosed viper (Vipera ammodytes ammodytes). Several residues in the C-terminal part of AtxA were found to be important in this interaction, particularly those in the region 115-119. In support of this finding, introduction of Y115, I116, R118 and N119, present in AtxA, into a weakly neurotoxic PLA2 from Russell's viper (Daboia russellii russellii) increased by sevenfold its binding affinity for CaM. Furthermore, two out of four peptides deduced from different regions of AtxA were able to compete with the toxin in binding to CaM. The nonapeptide showing the strongest inhibition was that comprising the AtxA region 115-119. This stretch contributes to a distinct hydrophobic patch within the region 107-125 in the C-terminal part of the molecule. This lacks any substantial helical structure and is surrounded by several basic residues, which may form a novel binding motif for CaM on the molecular surface of the PLA2 toxin.     

Structural basis for bile salt inhibition of pancreatic phospholipase A2

Bile salt interactions with phospholipid monolayers of fat emulsions are known to regulate the actions of gastrointestinal lipolytic enzymes in order to control the uptake of dietary fat. Specifically, on the lipid/aqueous interface of fat emulsions, the anionic portions of amphipathic bile salts have been thought to interact with and activate the enzyme group-IB phospholipase A2 (PLA2) derived from the pancreas. To explore this regulatory process, we have determined the crystal structures of the complexes of pancreatic PLA2 with the naturally occurring bile salts: cholate, glycocholate, taurocholate, glycochenodeoxycholate, and taurochenodeoxycholate. The five PLA2-bile salt complexes each result in a partly occluded active site, and the resulting ligand binding displays specific hydrogen bonding interactions and extensive hydrophobic packing. The amphipathic bile salts are bound to PLA2 with their polar hydroxyl and sulfate/carboxy groups oriented away from the enzyme's hydrophobic core. The impaired catalytic and interface binding functions implied by these structures provide a basis for the previous numerous observations of a biphasic dependence of the rate of PLA2 catalyzed hydrolysis of zwitterionic glycerophospholipids in the presence of bile salts. The rising or activation phase is consistent with enhanced binding and activation of the bound PLA2 by the bile salt induced anionic charge in a zwitterionic interface. The falling or inhibitory phase can be explained by the formation of a catalytically inert stoichiometric complex between PLA2 and any bile salts in which it forms a stable complex. The model provides new insight into the regulatory role that specific PLA2-bile salt interactions are likely to play in fat metabolism.     

Crystal structure of phospholipase A2 complex with the hydrolysis products of platelet activating factor: equilibrium binding of fatty acid and lysophospholipid-ether at the active site may be mutually exclusive

We have solved the 1.55 A crystal structure of the anion-assisted dimer of porcine pancreatic group IB phospholipase A2 (PLA2), complexed with the products of hydrolysis of the substrate platelet activating factor. The dimer contains five coplanar phosphate anions bound at the contact surface between the two PLA2 subunits. This structure parallels a previously reported anion-assisted dimer that mimics the tetrahedral intermediate of PLA2 bound to a substrate interface [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. The dimer structure has a molecule of the product acetate bound in subunit A and the other product 1-octadecyl-sn-glycero-3-phosphocholine (LPC-ether) to subunit B. Therefore, this structure is of the two individual product binary complexes and not of a ternary complex with both products in one active site of PLA2. Protein crystals with bound products were only obtained by cocrystallization starting from the initial substrate. In contrast, an alternate crystal form was obtained when PLA2 was cocrystallized with LPC-ether and succinate, and this crystal form did not contain bound products. The product bound structure has acetate positioned in the catalytic site of subunit A such that one of its oxygen atoms is located 3.5 A from the catalytic calcium. Likewise, a longer than typical Ca-to-Gly(32) carbonyl distance of 3.4 A results in a final Ca coordination that is four-coordinate and has distorted geometry. The other oxygen of acetate makes hydrogen bonds with N(delta)(1)-His(48), O(delta)(1)-Asp(49), and the catalytic assisting water (w7). In contrast, the glycerophosphocholine headgroup of LPC-ether in subunit B makes no contacts with calcium or with the catalytic residues His(48) or Asp(49). The tail of the LPC-ether is located near the active site pocket with the last nine carbons of the sn-1- acyl chain refined in two alternate conformations. The remaining atoms of the LPC-ether product have been modeled into the solvent channel but have their occupancies set to zero in the refined model due to disorder. Together, the crystallographic and equilibrium binding results with the two products show that the simultaneous binding of both the products in a single active site is not favored.     

Structure of protein having inhibitory disintegrin and leukotriene scavenging functions contained in single domain

The antihemostatic/antiangiogenic protein tablysin-15 is a member of the CAP (cysteine-rich secretory, antigen 5, and pathogenesis-related 1 protein) superfamily and has been shown to bind the integrins α(IIb)β(3) and α(V)β(3) by means of an Arg-Gly-Asp (RGD) tripeptide sequence. Here we describe the x-ray crystal structure of tablysin-15 and show that the RGD motif is located in a novel structural context. The motif itself is contained in a type II β-turn structure that is similar in its conformation to the RGD sequence of the cyclic pentapeptide cilengitide when bound to integrin α(V)β(3). The CAP domain also contains a hydrophobic channel that appears to bind a fatty acid molecule in the crystal structure after purification from Escherichia coli. After delipidation of the protein, tablysin-15 was found to bind proinflammatory cysteinyl leukotrienes with submicromolar affinities. The structure of the leukotriene E(4)-tablysin-15 complex shows that the ligand binds with the nonfunctionalized end of the fatty acid chain buried in the hydrophobic pocket, whereas the carboxylate end of the ligand binds forms hydrogen bond/salt bridge interactions with polar side chains at the channel entrance. Therefore, tablysin-15 functions as an inhibitor of integrin function and as an anti-inflammatory scavenger of eicosanoids.     

A model of the ACE2 structure and function as a SARS-CoV receptor

The angiotensin-converting enzyme 2 (ACE2) is an important regulator of the renin-angiotensin system and was very recently identified as a functional receptor for the SARS virus. The ACE2 sequence is similar (sequence identities 43% and 35%, and similarities 61% and 55%, respectively) to those of the testis-specific form of ACE (tACE) and the Drosophila homolog of ACE (AnCE). The high level of sequence similarity allowed us to build a robust homology model of the ACE2 structure with a root-mean-square deviation from the aligned crystal structures of tACE and AnCE less than 0.5A. A prominent feature of the model is a deep channel on the top of the molecule that contains the catalytic site. Negatively charged ridges surrounding the channel may provide a possible binding site for the positively charged receptor-binding domain (RBD) of the S-glycoprotein, which we recently identified [Biochem. Biophys. Res. Commun. 312 (2003) 1159]. Several distinct patches of hydrophobic residues at the ACE2 surface were noted at close proximity to the charged ridges that could contribute to binding. These results suggest a possible binding region for the SARS-CoV S-glycoprotein on ACE2 and could help in the design of experiments to further elucidate the structure and function of ACE2.