Evidence for dynamics in proteins as a mechanism for ligand dissociation

Abstract

Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or koff, should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into koff 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 Ki and koff, suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although kconf,forward is faster than koff, use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

“…Alternatively, the dissociation rates (k off ) are not governed by diffusion limits and can be significantly altered, providing access to a large range of binding affinities. The dissociation rates of transcriptional activators from DNA or small molecule ligands from proteins have been shown to govern the residence time of proteins and ligands in particular functional states and/or locations (41,42). It is possible that in this case the residence time of various TADs with the coactivator CBP is used to regulate the degree of transcriptional activation and/or histone acetyl transferase activity in cellular pathways.…”

Protein conformational dynamics dictate the binding affinity for a ligand

“…The role of conformational dynamics in a receptor-ligand interaction has been the subject of intensive studies 3,9,10,12,[16][17][18] . The receptor-ligand interaction process comprises two fundamental steps, namely, the binding and dissociation of a ligand.…”

“…With advances in experimental methods to investigate the protein dynamics, such as nuclear magnetic resonance, computational simulations and single-molecule techniques, evidence for the role of protein dynamics in ligand dissociation has been provided 9,10,19,20 . Recently, Carroll et al 3 analysed the dynamics at the binding site of DHFR for a series of inhibitors with different binding affinities through nuclear magnetic resonance relaxation dispersion. Interestingly, a set of residues near the binding site were observed to undergo a millisecondtimescale switching from ground to excited conformations.…”

“…Traditionally, ligand release has been explained using a simple diffusion model 3 . With advances in experimental methods to investigate the protein dynamics, such as nuclear magnetic resonance, computational simulations and single-molecule techniques, evidence for the role of protein dynamics in ligand dissociation has been provided 9,10,19,20 .…”

“…Many biochemical processes in living cells, however, occur in non-equilibrium conditions, and there has been a growing recognition that the binding rate (k 1 ) and dissociation rate (k À 1 ) of molecular interactions provide more decisive clues for understanding the cell physiology resulting from molecular interactions. For instance, the residence time of a ligand has a significant effect on the signal transduction, regulatory processes and drug responses [2][3][4][5] .…”