Novel Insights Into the Dynamic Conformational Transitions and Active Site Plasticity of Human Immunoregulatory Cathepsin S

ABSTRACT Cathepsin S (CatS), a cysteine protease, catalyzes the cleavage of immunoregulatory peptides and mediates tissue destruction in autoimmune and inflammatory diseases. Plasticity of its ligand binding site and mechanisms of dynamic transitions between different conformational states are critical in drug discovery; however, knowledge of its entire conformational landscape and transition mechanisms remains incomplete. Therefore, we investigated the atomic‐level interactions between active site cleft residues that contribute to its structural and functional plasticity. Here, we show that the hinge movement of side chains of Phe211, Phe70, and Tyr118, followed by side chain reorientation of active site residues and inter‐residue interactions, results in open or closed conformations, contributing to the plasticity of the S2 binding affinity hotspot pocket of CatS. Hinge movements of Phe211, Phe70, and Tyr118 regulate the space available in the S2 pocket, with Phe70 acting as a key regulator, thereby affecting small molecule binding in the active site cleft. Further, the non‐covalent interactions between active site residues during transitions between open and closed states lead to the formation of three distinct, dynamic, semi‐closed substates. The transition to the closed state can be blocked by a ligand that sterically hinders the hinge movement of Phe70 or Phe211. The cooperative, organized side chain rotation of Phe211, Phe70, and Tyr118, and subsequent emergence of non‐covalent interactions between the active site residues can influence the accommodation of ligands and their specificity. These novel findings might further aid the design of selective small molecule drugs targeting specific conformational states of the immunoregulatory and inflammatory/autoimmune disease target human CatS.