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Allostery governs Cdk2 activation and differential recognition of CDK inhibitors

Nature Chemical Biology volume 17, pages 456–464 (2021)Cite this article

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Abstract

Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).

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Fig. 1: Cyclin binding triggers a concerted conformational change in Cdk2.
Fig. 2: Monomeric Cdk2 dynamically samples a catalytically inactive Aloop-out state.
Fig. 3: Cdk2 inhibitors drive conformational shifts on binding.
Fig. 4: Tight binding of Cdk2 inhibitors arises from allosteric coupling with the cyclin subunit.
Fig. 5: A divergent hub controlling allosteric coupling in Cdk2.

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Data availability

The data that support the findings in this study are available within the paper (main and supplementary sections).

Code availability

The program used to analyze DEER data for this study, Venison, is available for download from https://github.com/thompsar/Venison.

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Acknowledgements

We thank M. Young for the Cdk2 and yeast CAK constructs, J. Endicott for the bovine cyclinA construct and S. Rubin for the RB771–928 construct. We thank J. Dalluge for assistance with mass spectrometry. This work was supported in part by grants from the National Institutes of Health (no. R01 GM121515, N.M.L.) and the National Institutes of Health Cancer Biology Training grant (no. T32 CA009138, A.M.) and Chemistry-Biology Interface Training grant (no. T32 GM132029, D.M.R.).

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Author notes

  1. These authors contributed equally: Abir Majumdar, David J. Burban.

Authors and Affiliations

  1. Department of Pharmacology and Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA

    Abir Majumdar, David J. Burban, Tiffany A. Engel & Nicholas M. Levinson

  2. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA

    Joseph M. Muretta, Andrew R. Thompson, Damien M. Rasmussen, Manu V. Subrahmanian, Gianluigi Veglia & David D. Thomas

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Contributions

N.M.L. and A.M. conceived the study. A.M. performed the DEER and FRET experiments. D.J.B. performed the NMR experiments. T.A.E. assisted with expression and purification of cyclinA and RB. J.M.M. supervised the global fitting analysis of the FRET data. A.R.T. assisted with the collection and analysis of DEER data. D.M.R. assisted with FRET experiments. M.V.S. assisted with NMR experiments. D.D.T., G.V. and N.M.L. provided overall guidance and supervision of the experiments. N.M.L. and A.M. wrote the manuscript.

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Correspondence to Nicholas M. Levinson.

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Extended data

Extended Data Fig. 1 Cdk2 inhibitors drive conformational shifts upon binding.

Aligned crystal structures of Cdk2 bound to dinaciclib and roscovitine (top), and structures of Cdk2:cyclinA bound to flavopiridol and ADP shown side by side (bottom). Hydrogen bonds are shown as yellow dashed lines, and structured water molecules as red spheres. The structure of Cdk2 bound to AZD5438 is shown in Supplementary Fig. 7.

Extended Data Fig. 2 A divergent hub controlling allosteric coupling in Cdk2.

a) KM values for phosphorylation of a short peptide substrate (left) and RB (right), measured for WT Cdk2, Cdk2cdk4hub and Cdk4. Values are mean ± S.E.M; n = 4 independent experiments. b) DEER data for the Cdk2cdk4hub mutant bound to cyclinA with and without addition of saturating peptide substrate and AMPPNP.

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Majumdar, A., Burban, D.J., Muretta, J.M. et al. Allostery governs Cdk2 activation and differential recognition of CDK inhibitors. Nat Chem Biol 17, 456–464 (2021). https://doi.org/10.1038/s41589-020-00725-y

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