Mapping the Binding Hotspots and Transient Binding Pockets on V-Domain Immunoglobulin Suppressor of T Cell Activation Protein Surface

V-domain immunoglobulin suppressor of T cell activation (VISTA), an inhibitory immune checkpoint present on both immune and tumor cells, has emerged as a highly promising target for cancer therapy due to its potential to overcome resistance encountered with existing immune checkpoint treatments. VSIG-3 is determined as an inhibitory ligand for VISTA, leading to the suppression of T cell proliferation. However, hotspots between VISTA/VSIG-3 protein-protein interaction remain ambiguous, mainly attributed to the lack of the structure of the VISTA/VSIG-3 complex. Therefore, in this study, in order to determine the energetic contributions of the interfacial residues on VISTA, we first constructed VISTA/VSIG-3 complex models by the protein docking method, followed by molecular dynamics simulations, binding free-energy decomposition, and alanine scanning. Results suggested that the putative hotspots in VISTA comprise residues His32, Tyr37, Thr35, Glu47, Val48, Gln49, Glu53, Arg54, Gln73, His122, and His126. Moreover, the distribution of the hotspots was clustered into two regions (hot regions I and II), and by using the TRAPP tool, transient subpockets within the hot regions were identified. Furthermore, conformational states of the binding pockets exhibiting druggability scores higher than those observed in the crystal structure were found. Overall, we hope that the findings outlined in this study can be used to facilitate the development of inhibitors targeting the VISTA/VSIG-3 immune checkpoint pathway in the future.