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Delineation of the allosteric mechanism of a cytidylyltransferase exhibiting negative cooperativity

Nature Structural Biology volume 8, pages 947–952 (2001)Cite this article

Abstract

The dimeric enzyme CTP:glycerol-3-phosphate cytidylyltransferase (GCT) displays strong negative cooperativity between the first and second binding of its substrate, CTP. Using NMR to study the allosteric mechanism of this enzyme, we observe widespread chemical shift changes for the individual CTP binding steps. Mapping these changes onto the molecular structure allowed the formulation of a detailed model of allosteric conformational change. Upon the second step of ligand binding, NMR experiments indicate an extensive loss of conformational exchange broadening of the backbone resonances of GCT. This suggests that a fraction of the free energy of negative cooperativity is entropic in origin.

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Figure 1: Structure and mechanism of GCT.
Figure 2: Changes in arginine NMR spectra observed upon titration of GCT with CTP.
Figure 3: Chemical shift mapping of CTP binding.
Figure 4: A model to describe the negative cooperativity of GCT.
Figure 5: Dynamics of GCT at the ms–μs timescale.

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Acknowledgements

This work was supported by grants from the National Institutes of Health. We thank C. Wilde for developing the protein purification procedure, and C. Weber and M.L. Ludwig for guiding us through the crystal structure and for stimulating discussions in general. We thank G. Yip for his contributions to the NMR relaxation experiments. The Keck Foundation, NIH and NSF are acknowledged for support of the 800 MHz system.

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Author notes

  1. Subramaniam Sanker

    Present address: Department of Molecular biology, NC2-119, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio, 44195, USA

Authors and Affiliations

  1. Biophysics Research Division, University of Michigan, 930 North University, Ann Arbor, 48109-1055, Michigan, USA

    Shawn Y. Stevens & Erik R.P. Zuiderweg

  2. Department of Biochemistry, University of Michigan, 4417 Medical Sciences Building I, Ann Arbor, 48109-0606, Michigan, USA

    Subramaniam Sanker, Claudia Kent & Erik R.P. Zuiderweg

  3. Department of Chemistry, University of Michigan, 930 North University, Ann Arbor, 48109-1055, Michigan, USA

    Erik R.P. Zuiderweg

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  1. Shawn Y. Stevens
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  4. Erik R.P. Zuiderweg
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Correspondence to Erik R.P. Zuiderweg.

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Stevens, S., Sanker, S., Kent, C. et al. Delineation of the allosteric mechanism of a cytidylyltransferase exhibiting negative cooperativity. Nat Struct Mol Biol 8, 947–952 (2001). https://doi.org/10.1038/nsb1101-947

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