BPS2026 – Capturing structural heterogeneity in enzymatic catalysis by adenylate kinase

Proteins perform their functions by adopting multiple transient conformations, which makes structural characterization challenging. Time-resolved X-ray solution scattering (TR-XSS) combined with caged compounds enables real-time observation of such conformational changes. However, delayed release from the photoprotective cage and ill-defined equilibrium states complicate interpretation. Using Escherichia coli adenylate kinase (AdK) as a model, we addressed these challenges with ensemble-based refinement guided by molecular dynamics simulations. Structural ensembles generated by molecular dynamics were compared with TR-XSS data to resolve heterogeneity caused by overlapping ATP release and domain motions. At equilibrium, adenylate kinase (AdK) favored intermediate conformations in which the binding domains were partially open rather than fully closed or open. Following ATP release, the ensembles shifted toward fully closed and fully open states, accompanied by partial unfolding. This behavior supports a cracking mechanism, where local unfolding facilitates large-scale conformational changes. Overall, the results show that delayed substrate release alters transition kinetics and highlight ensemble-based refinement as an effective approach for capturing rapid protein dynamics in solution.