Surface hydrophobic clusters modulate the folding stability and molecular recognition of the disintegrin jarastatin.

Abstract

Disintegrins are cysteine-rich proteins found in snake venoms. These proteins selectively bind to integrins, which play a key role in the regulation of many physiopathological processes. They are coreless proteins that display almost all hydrophobic residues on the protein surface. The exposed hydrophobic residues form surface clusters stabilized by the interaction with the hydrophilic residues in the vicinity and the hydration shell. In the present work, we aimed to determine the stability of surface hydrophobic clusters (SHCs) and their role in protein folding and biological activity. We used urea denaturation curves followed by ¹H and ¹⁵N chemical shifts to determine the free energy of unfolding (ΔG_F-U) and CLEANEX experiments to measure the water exchange rates of the surface amides (k_ex). The amides with higher local stability and protection from water exchange are those near or at the SHCs, which form a hydrophobic face. SHCs act as foldons, guiding oxidative folding and contributing to the formation of the disulfide bond framework, which is essential for establishing the concave shape and, ultimately, the binding cleft. On the opposite side of the protein are the residues with lower local stability and amides that exchange fast with water. This face coincides with the binding cleft of the protein to the αVβ3 integrin. Taken together, the present work established a correlation between protein hydration and the binding surface.