Analysis of Binding of Cobra Cardiotoxins to Heparin Reveals a New β-Sheet Heparin-binding Structural Motif

Alka A Vyas,Jiann-Jong Pan,Himatkumar V Patel,Kavita A Vyas,Chien-Min Chiang,You-Cheng Sheu,Jenn-Kang Hwang,Wen-Guey Wu

doi:10.1074/jbc.272.15.9661

Abstract

Heparin and heparan sulfate have recently been shown to bind to snake cardiotoxin (CTX) and to potentiate its penetration into phospholipid monolayer under physiological ionic conditions. Herein we analyze the heparin-binding domain of CTX using 10 CTXs from Taiwan and African cobra venom. We also performed computer modeling to obtain more information of the binding at molecular level. The results provide a molecular model for interaction of CTX-heparin complex where the cationic belt of the conserved residues on the concave surface of three finger beta-sheet polypeptides initiates ionic interaction with heparin-like molecules followed by specific binding of Lys residues near the tip of loop 2 of CTX. The dissociation constants of CTXs differ by as much as 4 orders of magnitude, ranging from approximately 140 microM for toxin gamma to approximately 20 nM for CTX M3, depending on the presence of Lys residues near the tip of loop 2. High affinity heparin binding becomes possible due to the presence of Arg-28, Lys-33, or the so-called consensus heparin binding sequence of XKKXXXKRX near the tip of the loop. The well defined three-finger loop structure of CTX provides an interesting template for the design of high affinity heparin-binding polypeptides with beta-sheet structure. The finding that several cobra CTXs and phospholipase A2 bind to heparin with different affinity may provide information on the synergistic action of the two venom proteins.

Highlights

Heparin and heparan sulfate (HS)1 belong to the glucosaminoglycan subclass of glycosaminoglycans (GAGs) and attract special attention because they exhibit greatest structural diversity [1, 2]
We have shown that titration of CTX A3 with heparin, monitored by change in ellipticity at 195 nm, and ϳ215 nm, yields a two-phase curve, initial decrease in ellipticity ϳ195 nm followed by recovery of the same ellipticity at higher GAG concentrations [10]
We have studied binding of 10 CTXs with LMW and HMW heparin by spectroscopic and affinity chromatographic techniques

Summary

Introduction

Heparin and heparan sulfate (HS) belong to the glucosaminoglycan subclass of glycosaminoglycans (GAGs) and attract special attention because they exhibit greatest structural diversity [1, 2]. Heparan sulfate proteoglycans are ubiquitous and abound at cell surfaces, wherefrom they control the entry or availability of approaching entities [3, 5] Numerous proteins, such as growth factors (for review, see Ref. 6), antithrombin III [7], phospholipase A2 (PLA2) [8, 9], among others, bind to heparin with dissociation constants ranging from ␮M to nM. Differing in some cases by as little as one residue, these naturally occurring variants present themselves as a series of readily available basic proteins that might allow establishment of trends underlying the interaction of basic ␤-sheet proteins with anionic carbohydrates of the cell surface This approach, though less specific than site-directed mutagenesis, can be directly applicable in understanding the toxicity of cobra venom. We analyzed heparin-induced conformational change of CTXs by circular dichroism (CD) spectro-

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Results

Conclusion