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Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme

Nature Structural & Molecular Biology volume 18, pages 288–294 (2011)Cite this article

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Abstract

Allostery has been studied for many decades, yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6′)-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homodimeric enzyme is modulated by opposing mechanisms. One follows a classical Koshland-Némethy-Filmer (KNF) paradigm, whereas the other follows a recently proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding and conformational changes represents a new way to govern energetic communication between binding sites.

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Figure 1: Schematic representation of homotropic allosteric models for a dimeric protein.
Figure 2: Temperature dependence of AAC(6′)-Ii binding thermodynamics and secondary structure.
Figure 3: Changes in AAC(6′)-Ii 800 MHz NMR spectra produced by AcCoA binding.
Figure 4: Analysis of NMR titration data.
Figure 5: Schematic representation of the allosteric binding model.
Figure 6: Dependence of the apparent cooperativity coefficient (αapp) on subunit instability (KU0 = [UF]/[FF]).

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Acknowledgements

The authors would like to thank G.D. Wright (McMaster University) for providing the AAC(6′)-Ii expression construct, as well as C.E. Ang and K. Shopsowitz for their assistance with data collection and analysis. This research was funded by operating grants from the Canadian Institutes of Health Research (CIHR) (K.A., A.K.M. and A.M.B.). L.A.F. and O.M.B. were supported through a CIHR training grant and CIHR scholarship, respectively. A.M.B. holds a Canada Research Chair in Structural Biology. A.M.B. and A.K.M. are supported by the Groupe de Recherche Axé sur la Structure des Protéines (GRASP). NMR experiments were recorded at the Québec/Eastern Canada High Field NMR Facility, supported by the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, the Ministère de la Recherche, de la Science et de la Technologie du Québec, and McGill University.

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  1. Department of Chemistry, McGill University, Montréal, Québec, Canada

    Lee A Freiburger, Karine Auclair & Anthony K Mittermaier

  2. Department of Biochemistry, McGill University, Montréal, Québec, Canada

    Oliver M Baettig & Albert M Berghuis

  3. Québec/Eastern Canada High Field NMR Facility, Montréal, Québec, Canada

    Tara Sprules

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  1. Lee A Freiburger
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L.A.F. and O.M.B. collected the data. L.A.F., O.M.B. and T.S. analyzed the data. A.K.M., K.A. and A.M.B. wrote the paper.

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Correspondence to Anthony K Mittermaier.

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Freiburger, L., Baettig, O., Sprules, T. et al. Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme. Nat Struct Mol Biol 18, 288–294 (2011). https://doi.org/10.1038/nsmb.1978

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