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

ABSTRACT 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.