Effect of P to A Mutation of the N-Terminal Residue Adjacent to the Rgd Motif on Rhodostomin: Importance of Dynamics in Integrin Recognition

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.

Dynamics and functional differences between dendroaspin and rhodostomin: Insights into protein scaffolds in integrin recognition

“…[35][36][37][38] In the present study, we showed that Den and Rho have different tertiary folds with similar 3D conformation for the RGD motif. However, their ability to inhibit the integrins avb3, a5b1, and aIIbb3 was different and to inhibit the integrin avb3 was significantly different.…”

“…It is known that the residues flanking the RGD motif of RGD-containing proteins affect their binding specificities and affinities on integrins. 27,36 Although Den and Rho have the same PRGDMP sequence in their RGD loop, they exhibited diverse specificity in inhibiting integrins. These results suggest that not only the flanking residues but also protein scaffold can affect the backbone dynamics of the RGD motif.…”

“…[6][7][8][9][10][11][12] Many studies have compared the binding affinity of RGD-containing peptide with their 3D conformations and backbone dynamics. 27,36,38,43 As shown in Table IV, the residues of the RGD loop of these proteins exhibited extensive flexibility on fast motion on the picasecond per nanosecond time scale. In general, proteins and peptides with a flexible RGD loop exhibited lower binding affinity and interacted with many integrins.…”

“…36,35,37,38 A summary of these values of the residues R, G, D, and M/S is presented in Table IV. Although both Den and Rho have a PRGDMP amino acid sequence in the loop, the backbone dynamics of this sequence are very different from those of the RGDM motif, except that their M residues exhibited very similar backbone dynamics.…”

“…27,36,43 Ninety-six-well microtiter plates (Costar; Corning) were coated with 100 lL of PBS buffer containing 200 lg/mL fibrinogen or 25 lg/mL FN and incubated overnight at 4 C. Nonspecific protein-binding sites were blocked by incubating each well with 200 lL of heat-denatured 1% BSA (Calbiochem) at room temperature for 1.5 h. The heat-denatured BSA was discarded, and each well was then washed twice with 200 lL of PBS.…”