LAUSR

Monobodies are synthetic antibody‐mimetic proteins that regulate enzyme functions through protein–protein interactions. In this study, we investigated the binding mechanisms of monobodies to adenylate kinase (Adk). Calorimetric and X‐ray crystallographic analyses revealed that CL‐1, a monobody specific for the CLOSED form of Adk, binds to the CORE domain of Adk in an enthalpy‐driven manner, forming several hydrogen bonds and a cation‐π interaction at the protein interface, without perturbing the Adk backbone. In contrast, OP‐4, an OPEN‐form‐specific monobody, exhibited entropy‐driven binding. 1H–15N 2D nuclear magnetic resonance (NMR), 31P‐NMR, and calorimetric studies revealed conformational perturbations to Adk by OP‐4, while substrate access remained intact. The different thermodynamic and structural effects between the monobodies highlight the diverse binding mechanisms among monobodies.