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Substrate-dependent switching of the allosteric binding mechanism of a dimeric enzyme

Nature Chemical Biology volume 10, pages 937–942 (2014)Cite this article

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Abstract

Enzyme activity is commonly controlled by allostery, where ligand binding at one site alters the activities of distant sites. Classical explanations for multisubunit proteins involve conformational transitions that are fundamentally deterministic. For example, in the Monod-Wyman-Changeaux (MWC) paradigm, conformational transitions occur simultaneously in all subunits. In the Koshland-Nemethy-Filmer (KNF) paradigm, conformational transitions only occur in ligand-bound subunits. In contrast, recent models predict conformational changes that are governed by probabilities rather than absolute rules. To better understand allostery at the molecular level, we applied a recently developed spectroscopic and calorimetric method to the interactions of a dimeric enzyme with two different ligands. We found that conformational transitions appear MWC-like for a ligand that binds at the dimer interface and KNF-like for a distal ligand. These results provide strong experimental support for probabilistic allosteric theory predictions that an enzyme can exhibit a mixture of MWC and KNF character, with the balance partly governed by subunit interface energies.

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Figure 1: Schematic representation of homotropic allosteric models for a dimeric protein.
Figure 2: Thermodynamics of paromomycin and AcCoA binding to AAC(6′)-Ii.
Figure 3: Structural analysis of ligand binding.
Figure 4: Joint NMR-ITC analysis of binding.
Figure 5: Allosteric properties of the EAM plotted as a function of ΔGint and ΔGtrans.

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Acknowledgements

The authors would like to thank G.D. Wright (McMaster University) for providing the AAC(6′)-Ii expression construct. This research was funded by operating grants from the Canadian Institutes of Health Research (CIHR MOP-89784) to K.A. and A.M. L.F. was supported through a CIHR training grant. A.B. holds a Canada Research Chair in Structural Biology. A.B. and A.M. are members of Groupe de Recherche Axé sur la Structure des Protéines (GRASP), which funds the NMR centre where the experiments were performed. NMR experiments were recorded at the Québec–Eastern Canada High Field NMR Facility, supported by McGill University and GRASP.

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Authors and Affiliations

  1. Department of Chemistry, McGill University, Montreal, Quebec, Canada

    Lee Freiburger, Teresa Miletti, Siqi Zhu, Karine Auclair & Anthony Mittermaier

  2. Department of Biochemistry, McGill University, Montreal, Quebec, Canada

    Oliver Baettig & Albert Berghuis

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  1. Lee Freiburger
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Contributions

A.M., K.A. and A.B. designed the experiments; L.F. collected and analyzed ITC and NMR data; T.M. analyzed NMR data; S.Z. collected and analyzed ITC data; O.B. analyzed X-ray crystallographic data; and A.M and L.F. wrote the paper.

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Correspondence to Anthony Mittermaier.

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Supplementary Results, Supplementary Tables 1 and 2, Supplementary Notes 1–3 and Supplementary Figures 1–4. (PDF 679 kb)

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Freiburger, L., Miletti, T., Zhu, S. et al. Substrate-dependent switching of the allosteric binding mechanism of a dimeric enzyme. Nat Chem Biol 10, 937–942 (2014). https://doi.org/10.1038/nchembio.1626

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