利用馬來蝮蛇蛇毒蛋白突變株研究在辨識組合蛋白αvβ3 和α5β1 的結構決定要素

Integrins are a family of cell surface receptors that mediate cell cellular adhesion, cell migration and signal transduction. It is known that integrins play important roles in the initiation and progression of many common diseases. Therefore, antagonism of integrins provides an approach for the treatment and prevention of these diseases. The RGD sequence is the cell attachment site of a large number of adhesive ECM, blood, and cell surface proteins and nearly half of 24 known integrins recognize this sequence in their adhesive ligands. Many studies have showed that the amino acid residues flanking the RGD motif of snake venom disintegrins control their binding specificity. Rhodostomin (Rho) is a potent integrin inhibitor and contains a PRGDMP sequence at positions of 48-53. Based on our results of cell adhesion analyses, we found that Rho mutants containing either ARGDGP or PRGDGP sequence can selectively inhibit integrin a v b 3 and Rho mutants containing ARGDXP sequence in the RGD loop have better inhibitory activity to integrin a 5 b 1. To understand structural determinants of integrins a v b 3 and a 5 b 1 recognition, we have determined 3D structures of these mutant proteins (ARGDMP, ARGDNP, ARGDGP and PRGDGP) by using NMR spectroscopy. Based on our structural analyses, the tertiary folds of wild-type and mutant proteins are the same. However, the backbone conformation of the RGD loop and the side-chain orientation of the D51 residue of mutant proteins (ARGDGP and PRGDGP) differ from those of wild-type Rho. Their structures are similar to our reported structure of the ARGDDL mutant, an integrin a v b 3-specific protein. These results suggest that these conformational differences may be responsible for their inhibitory selectivity to integrin a v b 3. In addition, model-free analyses of PRGDMP and ARGDMP proteins reveal that mutation of P48 to A caused a decrease in the order parameter (S2) of the R49 residue of the ARGDMP protein, mainly due to the decrease of R2. This difference may be responsible for the better inhibitory activity of Rho mutants containing ARGDXP sequence to integrin a 5 b 1. Using 3D structure of a v b 3 integrin as template, 3D model structures of integrins a IIb b 3 and a 5 b 1 were generated by using homology modeling. Docking integrins a IIb b 3, a v b 3, and a 5 b 1-specific Rho mutants to their receptors will be used to identify their possible interacting amino acids. Our findings suggest that the residues flanking the RGD motif of disintegrins regulate their structure, dynamics and function. These results will be used to design potent integrins a IIb b 3, a v b 3, and a 5 b 1-specific antagonists.