Binding mechanism of adenylate kinase-specific monobodies.

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.