Binding mechanism of adenylate kinase-specific monobodies

Abstract

Monobody, an antibody-mimetic protein, regulates enzyme functions via protein-protein interactions. This study examines the binding mechanisms of monobodies for adenylate kinase (Adk), focusing on thermodynamics and structural aspects. The calorimetric and X-ray crystallographic analyses for CL-1, a monobody specific to the CLOSED form of Adk, showed that CL-1 binds to the CORE domain in an enthalpy-driven manner, forming hydrogen bonds and a cation-π interaction at the interface with Adk. In contrast, OP-4, an OPEN-form-specific monobody, exhibited entropy-driven binding. The 1H-15N 2D nuclear magnetic resonance (NMR) and 31P-NMR studies showed the conformational perturbation to Adk by OP-4, while substrate access remains intact. The different thermodynamic and structural effects between CL-1 and OP-4 highlight the diversified binding mechanisms in monobodies.