Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbβ3 recognition

Rhodostomin (Rho) is a medium disintegrin containing a 48 PRGDMP motif. We here showed that Rho proteins with P48A, M52W, and P53N mutations can selectively inhibit integrin αIIbβ3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho 48 PRGDMP and 48 ARGDWN mutants in Pichia pastoris containing 65 P, 65 PR, 65 PRYH, 65 PRNGLYG, and 65 PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 ≥ α5β1, and the 48 ARGDWN- 65 PRNPWNG protein was the most selective integrin αIIbβ3 mutant. The 48 ARGDWN- 65 PRYH, 48 ARGDWN- 65 PRNGLYG, and 48 ARGDWN- 65 PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48 ARGDWN- 65 PRYH and 48 ARGDWN- 65 PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of 48 ARGDWN- 65 PRNPWNG. Based on the results of cell adhesion, platelet aggregation and the binding of fibrinogen to platelet inhibited by ARGDWN mutants, integrin αIIbβ3 bound differently to immobilized and soluble fibrinogen. NMR structural analyses of 48 ARGDWN- 65 PRYH, 48 ARGDWN- 65 PRNGLYG, and 48 ARGDWN- 65 PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48 ARGDWN interacted with H68 of 65 PRYH, L69 of 65 PRNGLYG, and N70 of 65 PRNPWNG, respectively. The docking of the 48 ARGDWN- 65 PRNPWNG mutant into integrin αIIbβ3 showed that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-π interaction with the β3 K125 residue. These results provide the first structural evidence that the interactions between the RGD loop and C-terminus of medium disintegrins depend on their amino acid sequences, resulting in their functional differences in the binding and selectivity of integrins.