Structural and Dynamic Differences of Rhodostomin, an RGD-containing Disintegrin, and its D51E Mutant
Yen-Chin Chen (陳彥青), Chiu-Yueh Chen, Ming-Jer Tang, Szecheng J. Lo, and Woei-Jer Chaung.
Department of Biochemistry, National Cheng Kung University Medical College, Tainan, Taiwan 701.
Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma. Rho consists of 68 amino acids including six disulfide bonds and an RGDMP sequence. Rho is a disintegrin that inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin aIIbbIII.of platelets. Rho produced in E. coli inhibited platelet aggregation with a KI of 263 nM. Although it is functional, Rho produced in E. coli is misfolded based on our nuclear magnetic resonance (NMR) studies. In order to correct the folding problem, Rho was expressed in Pichia pastoris. Rho produced in Pichia pastoris inhibits the platelet aggregation with a KI of 78 nM as potent as native Rho. However, its D51E mutant inhibits the platelet aggregation with a KI of 70 mM. In order to study the 900-fold decrease in the activity of D51E mutant, dichroism (CD) and NMR spectroscopy were used to determine the structural differences between Rho and its D51E mutant. Here we present the solution structures of Rho and its D51E mutant determined by heteronuclear multidimensional NMR spectroscopy and the hybrid distance geometry-dynamical simulated annealing method. Based on the solution structures of Rho and its D51E mutant, they have the same tertiary fold and the only difference is the distance between positive (R49) and negative residues (D51 or E51). This suggests that the length requirement may play the most important role in interaction between integrin and rhodostomin.