Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or k(off), should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) for a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase that vary by six orders of magnitude in binding affinity. NMR relaxation dispersion experiments revealed a common set of residues near the binding site that undergo a concerted, millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although k(conf,forward) is faster than k(off), use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.