Structural and Functional Differences of Rhodostomin and Echistatin in Integrin Recognition and Biological Implications.

Rhodostomin (Rho) and Echistatin (Ech) are RGD-containing disintegrins with different sizes, disulfide bond patterns, and amino acid sequences in their RGD loops and C-termini. Cell adhesion analyzes showed that Rho exhibited a 5.2-, 18.9-, 2.2-, and 1.7-fold lower inhibitory activity against integrins αvβ3, α5β1, αIIbβ3, and αvβ5 in comparison with those of Ech. In contrast, Rho exhibited an 8.8-fold higher activity than Ech in inhibiting integrin αvβ6. The swapping of Ech's RGD loop and C-terminal sequences into those of Rho cannot increase its integrins' inhibitory activities. Interestingly, the mutation of Ech into Rho's RGD loop PRGDMP sequence and C-terminal YH sequence caused an 8.2-fold higher activity in inhibiting integrin αvβ6. Structural analyzes of Rho and Ech showed that they have similar conformations in their RGD loop and different conformations in their C-terminal regions. Molecular docking found that not only the RGD loop but also the C-terminal region of Rho and Ech interacted with integrins, showing that the C-terminal region is also important for integrin recognition. The docking of Rho into integrin αvβ6 showed that the C-terminal H68 residue of Rho interacted with D129 of β6. In contrast, the docking of Ech into integrin α5β1 showed that the C-terminal H44 residue of Ech interacted with Q191 of β1. Ech exhibited 78.5- and 10.9-fold higher activities in inhibiting HUVEC proliferation and A375 melanoma cell migration than those of Rho. These findings demonstrate that the disulfide bond pattern, RGD loop, and C-terminal region of disintegrins may cause their functional differences. The functional and structural differences between Rho and Ech support their potential as scaffolds to design drugs targeting their respective integrins.