Abstract
Rhodostomin (Rho) is an RGD protein that specifically inhibits integrins. We found that Rho mutants with the P48A mutation 4.4–11.5 times more actively inhibited integrin α5β1. Structural analysis showed that they have a similar 3D conformation for the RGD loop. Docking analysis also showed no difference between their interactions with integrin α5β1. However, the backbone dynamics of RGD residues were different. The values of the R2 relaxation parameter for Rho residues R49 and D51 were 39% and 54% higher than those of the P48A mutant, which caused differences in S2, Rex, and τe. The S2 values of the P48A mutant residues R49, G50, and D51 were 29%, 14%, and 28% lower than those of Rho. The Rex values of Rho residues R49 and D51 were 0.91 s−1 and 1.42 s−1; however, no Rex was found for those of the P48A mutant. The τe values of Rho residues R49 and D51 were 9.5 and 5.1 times lower than those of P48A mutant. Mutational study showed that integrin α5β1 prefers its ligands to contain (G/A)RGD but not PRGD sequences for binding. These results demonstrate that the N-terminal proline residue adjacent to the RGD motif affect its function and dynamics, which suggests that the dynamic properties of the RGD motif may be important in Rho's interaction with integrin α5β1.
Highlights
The tripeptide sequence Arg-Gly-Asp (RGD) is the consensus sequence of many adhesive proteins, such as fibronectin, fibrinogen, vitronectin, and von Willebrand factor [1,2,3]
These results demonstrate that the N-terminal proline residue adjacent to the RGD motif affect its function and dynamics, which suggests that the dynamic properties of the RGD motif may be important in Rho’s interaction with integrin a5b1
Rho and its P48A mutant inhibited K562-cell adhesion to immobilized fibronectin with the IC50 values of 256.8687.5 and 59.0628.4 nM, respectively. In contrast to their inhibition of integrins aIIbb3 and avb3, P48A mutant showed a 4.4-fold increase in its inhibition of integrin a5b1. These results showed that the N-terminal alanine residue adjacent to the RGD motif increased its inhibition of integrin a5b1, but not of integrins aIIbb3 and avb3
Summary
The tripeptide sequence Arg-Gly-Asp (RGD) is the consensus sequence of many adhesive proteins, such as fibronectin, fibrinogen, vitronectin, and von Willebrand factor [1,2,3]. Disintegrins are the peptides found in snake venoms of the viper family and mainly inhibit the functions of b1- and b3-associated integrins. They were first identified as inhibitors of integrin aIIbb and were subsequently shown to bind with high affinity to other integrins and to block the interaction of integrins with RGD-containing proteins. They contain 47–84 amino acids with 4–7 disulfide bonds. The R and D sidechains in a flexible loop do not interact but nearly oppose each other by 180u
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