Effect of D to E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: Importance of the interactions between the D residue and integrin

Rhodostomin (Rho) is a snake venom protein containing an RGD motif that specifically inhibits the integrin-binding function. Rho produced in Pichia pastoris inhibits platelet aggregation with a K _I of 78 nM as potent as native Rho. In contrast, its D51E mutant inhibits platelet aggregation with a K _I of 49 μM. Structural analysis of Rho and its D51E mutant showed that they have the same tertiary fold with three twostranded antiparallel β-sheets. There are no structural backbone differences between the RG[D/E] loop which extends outward from the protein core and the RG[D/E] sequence at its apex in a four-residue RG[D/E]M type I turn. Two minor differences between Rho and its D51E mutant were only found from their backbone dynamics and 3D structures. The R ₂ value of E51 is 13% higher than that of the D51 residue. A difference in the charge separation of 1.76 Å was found between the sidechains of positive (R49) and negative residues (D51 or E51).The docking of Rho into integrin αvβ3 showed that the backbone amide and carbonyl groups of the D51 residue of Rho were formed hydrogen bonds with the integrin residues R216 and R214, respectively. In contrast, these hydrogen bonds were absent in the D51E mutantintegrin complex. Our findings suggest that the interactions between both the sidechain and backbone of the D residue of RGD-containing ligands and integrin are important for their binding.