LAUSR

While extremely important for relating the protein structure to its biological function, determination of the protein conformational transition pathway upon ligand binding is made difficult due to the transient nature of intermediates, a large and rugged conformational space, and coupling between protein dynamics and ligand-protein interactions. Existing methods that rely on prior knowledge of the bound (holo) state structure are restrictive. A second concern relates to the correspondence of intermediates obtained to the metastable states on the apo → holo transition pathway. Here, we have taken the protein apo structure and ligand-binding site as only inputs and combined an elastic network model (ENM) representation of the protein Hamiltonian with linear response theory (LRT) for protein-ligand interactions to identify the set of slow normal modes of protein vibrations that have a high overlap with the direction of the protein conformational change. The structural displacement along the chosen direction was performed using excited normal modes molecular dynamics (MDeNM) simulations rather than by the direct use of LRT. Herein, the MDeNM excitation velocity was optimized on-the-fly on the basis of its coupling to protein dynamics and ligand-protein interactions. Thus, a determined set of structures was validated against crystallographic and simulation data on four protein-ligand systems, namely, adenylate kinase-di(adenosine-5')pentaphosphate, ribose binding protein-β-d-ribopyranose, DNA β-glucosyltransferase-uridine-5'-diphosphate, and G-protein α subunit-guanosine-5'-triphosphate, which present important differences in protein conformational heterogeneity, ligand binding mechanism, viz. induced-fit or conformational selection, extent, and nonlinearity in protein conformational changes upon ligand binding, and presence of allosteric effects. The obtained set of intermediates was used as an input to path metadynamics simulations to obtain the free energy profile for the apo → holo transition.