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Evidence for dynamics in proteins as a mechanism for ligand dissociation

Nature Chemical Biology volume 8, pages 246–252 (2012)Cite this article

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Abstract

Signal transduction, regulatory processes and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times (1/koff) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into koff involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude. 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 the binding affinity 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 the forward rate of conformational exchange, kconf,forward, is faster than koff, the use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.

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Figure 1: The series of reduced-affinity and previously characterized antifolates.
Figure 2: High-resolution crystal structures for the series.
Figure 3: Slow timescale dynamics for the reduced-affinity inhibitor series.
Figure 4: Internal motions vary with Ki and koff.
Figure 5: Antifolate consensus sites sample a structurally similar excited state.

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Acknowledgements

The authors thank G. Young (University of North Carolina at Chapel Hill Biomolecular NMR Facility) and K. Koshlap (University of North Carolina at Chapel Hill Eshelman School of Pharmacy NMR Facility) for their excellent technical assistance. We also acknowledge H. Kohn for assistance in assigning and interpreting the small-molecule NMR spectra as well as N. Kett for chiral resolution of 3 and the lab of P. Guengerich for use of their stopped-flow fluorimeter. M.J.C. gratefully acknowledges predoctoral fellowships from the ACS Division of Medicinal Chemistry (supported by Pfizer Global R&D), the American Foundation for Pharmaceutical Education (supported by the Rho Chi Society and Schering-Plough) and the Graduate School at UNC. This work was funded by US National Institutes of Health grant GM083059 to A.L.L.

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Authors and Affiliations

  1. Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Mary J Carroll, Anna V Gromova, Scott F Singleton & Andrew L Lee

  2. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Randall V Mauldin, Edward J Collins & Andrew L Lee

  3. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Edward J Collins

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  1. Mary J Carroll
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M.J.C., A.L.L. and S.F.S. designed research; M.J.C., R.V.M., A.V.G. and E.J.C. performed research; M.J.C., R.V.M., A.V.G., S.F.S., E.J.C. and A.L.L. analyzed data; and M.J.C. and A.L.L. wrote the paper.

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Correspondence to Andrew L Lee.

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Carroll, M., Mauldin, R., Gromova, A. et al. Evidence for dynamics in proteins as a mechanism for ligand dissociation. Nat Chem Biol 8, 246–252 (2012). https://doi.org/10.1038/nchembio.769

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