Structural Basis of Phospholipase A₂ Inhibition for the Synthesis of Prostaglandins by the Plant Alkaloid Aristolochic Acid from a 1.7 Å Crystal Structure^,

Abstract

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).

Mass spectrometry analysis of the phospholipase A2 activity of snake pre‐synaptic neurotoxins in cultured neurons

“…4). Following pre-incubation with the generic cellular PLA 2 inhibitor AA (Vishwanath et al 1988;Rosenthal et al 1989;Chandra et al 2002), no reduction in the LysoPLs production by Tetx was detected, whereas a small decrease (significative with p < 0.05) was observed in the case of Ntx. This latter observation might be because of the slight inhibition of the Ntx enzymatic activity itself by AA as detected by performing an in vitro PLA 2 assay (not shown).…”

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

“…It may be emphasized that the peptide FLSYK forms a larger number of interactions with PLA 2 as compared with those observed in other complexes formed between PLA 2 and synthetic/natural inhibitors reported earlier (10,11,(13)(14)(15)(16)(37)(38)(39)(40)(41) making it to be the strongest inhibitor known so far against PLA 2 . Its dissociation constant, K d was found to be 3.57 Ϯ 0.05 ϫ 10 Ϫ9 M. Recently, molecular modeling studies showing the binding of FLSYK to human sPLA 2 -IIA were also reported (19).…”

“…This was reflected in the form of a very low dissociation constant, K d at 3.57 Ϯ 0.05 ϫ 10 Ϫ9 M for the PLA 2 -FLSYK complex. The corresponding dissociation constants for the complexes with ␣Ϫtocopherol (39), aristolochic acid (40), and LAIYS (41) were found to be 1.63 Ϯ 0.04 ϫ 10 Ϫ6 M, 1.37 Ϯ 0.05 ϫ 10 Ϫ6 M, and 9.74 Ϯ 0.03 ϫ 10 Ϫ7 M, respectively.…”

“…The structure of PLA 2 from Daboia russelli pulchella has been studied in its native form (24) and in the complexed forms with natural substances such as ␣-tocopherol (39) and aristolochic acid (40), and with a designed peptide inhibitor Leu-Ala-IleTyr-Ser (LAIYS) (41). In all these structures, PLA 2 exists in the form of an asymmetric dimer.…”

Design of specific peptide inhibitors of phospholipase A2: structure of a complex formed between Russell's viper phospholipase A2and a designed peptide Leu-Ala-Ile-Tyr-Ser (LAIYS)

“…The ability of PLA 2 s to produce substrates for the generation of in¯am-matory lipid mediators in the process of tissue injury and rheumatoid arthritis (Wery et al, 1991) makes this speci®c class of phospholipases very important targets for the design of speci®c drugs against in¯ammation. We have previously reported complexes of phospholipase A 2 with vitamin E (Chandra et al, 2002a) and aristolochic acid (Chandra et al, 2002b) in which OH groups from these inhibitors formed hydrogen bonds with Asp49 O 1 and His48 N 1 simulta-…”