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Directional conformer exchange in dihydrofolate reductase revealed by single-molecule nanopore recordings

Nature Chemistry volume 12, pages 481–488 (2020)Cite this article

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Abstract

Conformational heterogeneity is emerging as a defining characteristic of enzyme function. However, understanding the role of protein conformations requires their thermodynamic and kinetic characterization at the single-molecule level, which remains extremely challenging. Here we report the ligand-induced conformational changes of dihydrofolate reductase (DHFR) by measuring the modulation of the nanopore currents. The long observation time of the electrical recordings enabled the detection of rare conformational transitions hidden in ensemble measurements. We show that DHFR exists in at least four ground-state configurations or conformers with different affinities for its ligands. Unliganded DHFR adopted low-affinity conformers, whereas the binding of substrates promoted the switch to the high-affinity conformer. Conversion between the conformers was accelerated by molecules that stabilized the transition state of DHFR, which suggests that the reaction lowers the energy barrier for conformer exchange and thus facilitates product release. This mechanism might be a general feature in enzymatic reactions affected by product inhibition or when the release of products is the rate-limiting step.

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Fig. 1: DHFR inside the ClyA-AS nanopore.
Fig. 2: Binding of NADPH and folate to DHFRtag reveals the different DHFR conformers.
Fig. 3: DHFR exist in four different DHFR conformers.
Fig. 4: DHFR conformer distribution depends on the reaction conditions.
Fig. 5: Exchange of conformers at the transition state.

Data availability

Raw data are provided in Supplementary Data 1. All other data are available from the authors upon reasonable request.

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Acknowledgements

A.B. is funded by a PhD grant from the Agency for Innovation by Science and Technology (IWT) Flanders. N.S.G. was funded by European Research Council (ERC).

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Authors and Affiliations

  1. Groningen Biomolecular Sciences & Biotechnology (GBB) Institute, University of Groningen, Groningen, The Netherlands

    Nicole Stéphanie Galenkamp & Giovanni Maglia

  2. Department of Chemistry, KU Leuven, Leuven, Belgium

    Annemie Biesemans & Giovanni Maglia

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N.S.G. performed the experiments and analysed the data presented in the manuscript. A.B. cloned the DNA and performed the preliminary electrophysiological experiments. N.S.G. and G.M. wrote the manuscript. G.M. supervised the project. All the authors discussed the results and commented on the manuscript.

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Correspondence to Giovanni Maglia.

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Supplementary Information

Additional results and discussion, extended materials and methods, Supplementary Figs. 1–8, Tables 1–5 and references.

Supplementary Data 1

A .zip file containing raw unfiltered traces used in the manuscript.

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Galenkamp, N.S., Biesemans, A. & Maglia, G. Directional conformer exchange in dihydrofolate reductase revealed by single-molecule nanopore recordings. Nat. Chem. 12, 481–488 (2020). https://doi.org/10.1038/s41557-020-0437-0

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