NMR insights into the structure, dynamics, and hydration mechanisms driving disintegrin–integrin recognition.

Disintegrins are a family of small cysteine-rich proteins from snake venoms that act as potent and selective inhibitors of integrins, a class of cell-surface receptors essential for adhesion, migration, and signaling. Owing to their ability to modulate integrin activity, disintegrins are not only valuable molecular tools to probe integrin biology but also promising leads for therapeutic intervention in thrombosis, cancer, and viral infections. Among them, Jarastatin (rJast) displays high affinity for the integrin αVβ3, a receptor overexpressed in tumor cells and directly implicated in cell proliferation and metastasis. Despite their biomedical relevance, the molecular details underlying disintegrin–integrin recognition and inhibition remain poorly understood. To gain insights into the mechanism of action of disintegrins, we combined structural and dynamic approaches, integrating solution NMR spectroscopy, molecular docking, and molecular dynamics simulations. We determined the structure of rJast, which adopts the canonical disintegrin fold and exhibits conformational plasticity across multiple timescales. Our findings show that αVβ3 recognition involves not only the RGD loop but also the N-terminal domain, with flexibility being a key determinant for high-affinity binding. To further dissect the molecular determinants of stability and hydration, we employed urea denaturation curves monitored by ¹H/¹⁵N chemical shifts to calculate free energies of unfolding (ΔG_F–U), together with CLEANEX experiments to measure water exchange rates (k_ex). Residues with higher stability (ΔG_F–U > 20 kJ/mol) and low water permeability cluster around a hydrophobic surface stabilized by disulfide bonds and hydrophobic contacts. Conversely, less stable and more solvent-permeable residues map onto the αVβ3-binding cleft. This direct correlation between hydration dynamics and integrin recognition unveils the role of a structurally stable hydrophobic scaffold in supporting the flexible binding interface. Together, these results provide a structural and dynamic framework for understanding how disintegrins inhibit integrins and open new perspectives for exploiting their mechanism of action in therapeutic development.