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 [1]. In this study, we expressed the disintegrin jarastatin (rJAST) Komagataella phaffii, assigned [2] and solved the structure, backbone dynamics, and determine the binding mechanism to integrin αVβ3. We measured the amide backbone relaxation parameters 15N (15N R1, 15N R2, and 1H-15N heteronuclear NOE) and the 15N Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD). We observed that dynamics play an important role in the interaction. Many residues at the interface of interaction with integrin αVβ3 are undergoing conformational exchange or thermal flexibility. We observed the involvement of the N-terminal domain in the interaction along with the C-terminal domain containing the loop where the RGD (Arg-Gly-Asp) binding motif is located. Finally, we performed NMR-data-derived docking (HADDOCK 2.4) of the rJAST and αVβ3 complex, using information from the literature and our experimental CSP and dynamics data. To obtain information about the stability of the rJAST/αVβ3 complex, the NMR structures of rJAST (PDB: 8S9E) and αVβ3 ectodomain (PDB: 4MMX) were used as initial structures for molecular dynamics simulations (MD). Three replicas of a 100 ns MD simulation of the rJast/αVβ3 complex were performed, starting from the lowest energy structure of the docking model, monitoring the root mean square deviation (RMSD) in function of time. The RMSD remained below 0.6 nm in all replicas, indicating that the rJast/αVβ3 complex is stable throughout the trajectories, with the N-terminal and C-terminal domains remaining associated with the integrin. The stable interactions between rJast and αVβ3 during the MD simulation were also analyzed.
Acknowledgments: FAPERJ, CNPq, CAPES, CNRMN