Dynamics connect substrate recognition to catalysis in protein kinase A

A Corrigendum to this article was published on 18 April 2011

This article has been updated

Abstract

Atomic resolution studies of protein kinases have traditionally been carried out in the inhibitory state, limiting our current knowledge on the mechanisms of substrate recognition and catalysis. Using NMR, X-ray crystallography and thermodynamic measurements, we analyzed the substrate recognition process of cAMP-dependent protein kinase (PKA), finding that entropy and protein dynamics play a prominent role. The nucleotide acts as a dynamic and allosteric activator by coupling the two lobes of apo PKA, enhancing the enzyme dynamics synchronously and priming it for catalysis. The formation of the ternary complex is entropically driven, and NMR spin relaxation data reveal that both substrate and PKA are dynamic in the closed state. Our results show that the enzyme toggles between open and closed states, which indicates that a conformational selection rather than an induced-fit mechanism governs substrate recognition.

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Figure 1: X-ray crystal structure of the PKA-C ternary complex containing AMP-PNP and PLN1–19.
Figure 2: Details of the interaction of isotopically labeled PLN1–20 with PKA-C.
Figure 3: Mapping of the backbone amide dynamics of PKA-C from the apo to ternary complex.
Figure 4: Opening and closing of the enzyme active site cleft.
Figure 5: Model for the mechanism of the formation of a catalytically competent ternary complex.

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  • 18 April 2011

    In the version of this article initially published, the authors incorrectly acknowledged funding from the US National Institutes of Health, GM64742. The correct grant number should be GM072701. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by the US National Institutes of Health (GM072701 and HL080081 to G.V. and GM19301 to S.S.T.). NMR data were collected at the National Magnetic Resonance Facility at Madison (NMRFAM) (US National Institutes of Health: P41RR02301, P41GM66326, RR02781 and RR08438; US National Science Foundation: DMB-8415048, OIA-9977486 and BIR-9214394) and the University of Minnesota NMR Facility (US National Science Foundation BIR-961477). We thank J.P. Loria (Yale University) for providing the TROSY Hahn echo pulse sequence, and we would also like to thank J.P. Loria, E.E. Metcalfe and G. Melacini for critical analysis of the paper.

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L.R.M., C.C., T.Y., M.T., A.K., S.S.T. and G.V. designed experiments, analyzed data and wrote the paper, and L.R.M., C.C., T.Y. and M.T. performed experiments.

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Correspondence to Susan S Taylor or Gianluigi Veglia.

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Supplementary Methods, Supplementary Results, Supplementary Figures 1–8 and Supplementary Tables 1 & 2 (PDF 5952 kb)

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Masterson, L., Cheng, C., Yu, T. et al. Dynamics connect substrate recognition to catalysis in protein kinase A. Nat Chem Biol 6, 821–828 (2010). https://doi.org/10.1038/nchembio.452

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