Crystal Structure of a Complex Formed between a Snake Venom Phospholipase A2 and a Potent Peptide Inhibitor Phe-Leu-Ser-Tyr-Lys at 1.8 Å Resolution

Abstract

Phospholipase A 2 is an important enzyme involved in the production of prostaglandins and their related compounds causing inflammatory disorders. Among the several peptides tested, the peptide Phe-Leu-Ser-Tyr-Lys (FLSYK) showed the highest inhibition. The dissociation constant (K d ) for this peptide was calculated to be 3.57 ؎ 0.05 ؋ 10 ؊9 M. In order to further improve the degree of inhibition of phospholipase A 2 , a complex between Russells viper snake venom phospholipase A 2 and a peptide inhibitor FLSYK was crystallized, and its structure was determined by crystallographic methods and refined to an R-factor of 0.205 at 1.8 Å resolution. Phospolipase A 2 (EC 3.1.1.4, PLA 2 ) 1 enzyme specifically cleaves the sn-2 acyl ester bond of phospholipids (1). These enzymes were first described as components of snake venoms and were later identified in mammals. PLA 2 s of mammalian origin were found in the pancreas (2), blood platelets, macrophages, and neutrophils (3, 4). These are also found in fluids derived from patients with inflammatory conditions (5, 6) and are induced in several cell types in response to inflammatory stimuli (4). It is understood that the concentrations of PLA 2 enzymes in serum and tissues correlate with the disease severity in several immune-mediated inflammatory pathologies in humans. These are also associated with the onset of rheumatoid arthritis (7, 8) and septic shock (5, 9). Therefore, it is of great interest to develop specific inhibitors for each of these enzymes. Several crystal structures of the tetrahedral mimic inhibitors with an sn-2-phosphate, substituent, L-1-(O-octyl-2-(heptyl phosphonyl)-sn-glycero-3-phosphoethanolamine, complexed with secreted PLA 2 from Chinese cobra venom (10), bee venom (11), and inflammatory exudates (12) have been reported. Also the crystal structure of an sn-2-acyl-amino analogue of phospholipids complexed with the engineered (without the residues of the surface 62-66) porcine PLA 2 (13) has been determined. The crystal structures of the complexes of bovine pancreatic PLA 2 with an inhibitor n-dodecyl-phosphorylcholine (14) and human synovial PLA 2 with an acyl amino analogue of phospholipids (15) are also of some interest. Recently, a series of indole inhibitors of human secretory PLA 2 were developed (16). Further, there is an example of a natural inhibition (17) where specific interactions from one molecule of a heterodimer acts as a natural inhibitor for the other. The synthetic peptides derived from the primary sequence of the PLA 2 itself were also studied as specific inhibitors (18,19). The kinetic studies (18, 19) support their high inhibitory potencies. These peptide inhibitors were stable against acyl esterase as well as phospholipase A 1 , C, and D. Therefore, they were suited for in vivo studies where phospholipids may be more readily metabolized. As part of the program on structure-based rational design of specific potent inhibitors using a model snake venom phospholipase A 2 , we have synthesized a number of peptides (20). One of these ...

Simultaneous inhibition of anti‐coagulation and inflammation: crystal structure of phospholipase A2 complexed with indomethacin at 1.4 Å resolution reveals the presence of the new common ligand‐binding site

“…There have also been extensive investigations to understand their (www.interscience.wiley.com) DOI:10.1002/jmr.960 catalytic actions and various ligands have also been designed to inhibit their enzymatic function. The structures of several complexes of PLA 2 with natural compounds (Chandra et al, 2002a(Chandra et al, , 2002dSingh et al, 2006a), substrate analogues (Scott et al, 1990;Thunnissen et al, 1990;Scott et al, 1991;Sekar et al, 1997;Epstein et al, 2001;Pan et al, 2001;Hansford et al, 2003) and with other designed synthetic compounds (Schevitz et al, 1995;Chandra et al, 2002bChandra et al, , 2002cSingh et al, 2003) have also been reported. The anti-inflammatory response of non-steroidal anti-inflammatory drugs (NSAIDs) has been attributed to their binding to PLA 2 (Singh et al, , 2006bJabeen et al, 2005) and COX enzymes Gierse et al, 1999).…”

Structural analysis of a group III Glu62-phospholipase A2 from the scorpion, Mesobuthus tamulus: Targeting and reversible inhibition by native peptides

“…Apart from a large number of van der Waals interactions, the peptide ligand (P) makes hydrogen bonds with certain residues in the channel. The hydrogen bonded interactions include Ser10 O␥· · ·Ala(P1) In the same study, it has been established that a peptide inhibitor consisting of hydrophobic residues and a basic residue at the C-terminus is an ideal design of a PLA 2 inhibitor [37]. Therefore the native peptide inhibition analysis in this study is totally in accordance with the crystal structure data.…”

“…This type of pentapeptide is reminiscent of native derived pentapeptides that have been shown to effectively inhibit PLA 2 [36,37]. With this background we sought to explain the necessity of the pentapeptide in the MtsPLA 2 .…”

Structural Elements of Ligand Recognition Site in Secretory Phospholipase A2 and Structure-Based Design of Specific Inhibitors

“…The structures of several complexes of PLA 2 with natural compounds [29][30][31], substrate analogues [17,32], indole derivatives [37][38], non-steroidal anti-inflammatory drugs (NSAIDs) [33][34][35] and with designed peptides [39][40] have been investigated. The substrate-binding site in PLA 2 is a part of the hydrophobic channel.…”

“…Several structures of PLA 2 complexes with natural compounds [29][30][31], substrate analogues [17,32], nonsteroidal anti-inflammatory drugs (NSAIDs) [33][34][35] and other designed synthetic compounds including peptides [36,[39][40] have been reported. These studies have led to a complete identification of the structural elements of ligand recognition in the hydrophobic channel of PLA 2 , thus paving the way to further improving the quality of inhibitors using structure-based design approach.…”