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Energetics of ångström-scale conformational changes in an RCK domain of the MthK K+ channel

Nature Structural & Molecular Biology volume 26, pages 808–815 (2019)Cite this article

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Abstract

Allosteric proteins transition among different conformational states in a ligand-dependent manner. Upon resolution of a protein’s individual states, one can determine the probabilities of these states, thereby dissecting the energetic mechanisms underlying their conformational changes. Here we examine individual regulator of conductance to K+ (RCK) domains that form the regulatory module of the Ca2+-activated MthK channel. Each domain adopts multiple conformational states differing on an ångström scale. The probabilities of these different states of the domain, assessed in different Ca2+ concentrations, allowed us to fully determine a six-state model that is minimally required to account for the energetic characteristics of the Ca2+-dependent conformational changes of an RCK domain. From the energetics of this domain, we deduced, in the framework of statistical mechanics, an analytic model that quantitatively predicts the experimentally observed Ca2+ dependence of the channel’s open probability.

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Fig. 1: Polarized fluorescence intensities, and calculated Itot, θ, φ and Ω at two Ca2+ concentrations.
Fig. 2: Comparison of θ and Ω angles determined from single-molecule fluorescence polarization measurements with those from existing crystal structures.
Fig. 3: Ca2+ dependence of state probabilities and state diagram of an energetic model of RCK.
Fig. 4: Model of the regulatory module in two configurations.
Fig. 5: Model for the whole MthK channel.

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Data and materials described here will be made available upon reasonable request.

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Acknowledgements

We thank Y. Zhou for technical support; Y. Jiang and R. MacKinnon for providing the cDNA of MthK; V. Pau and B. Rothberg for sharing their published data for comparison; and P. De Weer, T. Hoshi and B. Salzberg for critiques of our manuscript at different stages of its development. This study was supported by the grant no. GM055560 from the National Institute of General Medical Sciences of the National Institutes of Health to Z.L.

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  1. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    John H. Lewis & Zhe Lu

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  1. John H. Lewis
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Contributions

J.H.L. and Z.L. designed the study; J.H.L. performed experiments, developed analytical tools, and analyzed the data, with the input from Z.L.; J.H.L. and Z.L. interpreted the results and wrote the manuscript.

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Correspondence to Zhe Lu.

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Peer review information: Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Crystal structure of the regulatory module of the MthK channel.

Ca2+ ions bind at three different sites, C1, C2 and C3, in individual RCK domains (PDB: 3RBZ).

Supplementary Figure 2 Comparison of models assuming different RCK conformations underlying the channel’s open states.

The three po-[Ca2+] curves calculated by assuming all-S2 species (blue, po), all-S1 (orange, PoS1), or both all-S1 and all-S2 species together (black, poS1,S2) underlie the open states. The black and blue curves are too close to be readily distinguished. For simplicity, gK was not included.

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Supplementary Figures 1–2, Supplementary Tables 1–2, Supplementary Notes 1–4

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Lewis, J.H., Lu, Z. Energetics of ångström-scale conformational changes in an RCK domain of the MthK K+ channel. Nat Struct Mol Biol 26, 808–815 (2019). https://doi.org/10.1038/s41594-019-0275-1

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