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Constructing ion channels from water-soluble α-helical barrels

Abstract

The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide–lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.

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Fig. 1: Computational design and characterization of water-soluble αHBs with solvated lumens.
Fig. 2: Engineering and characterization of transmembrane ion-channel peptides.
Fig. 3: LCP crystal structure of K2-CCTM-VbIc and modelling of alternate states.
Fig. 4: CCTM-VbIc channels in DIBs.

Data availability

X-ray crystal structures have been submitted to the RCSB Protein Data Bank with accession codes 6YAZ, 6YB0, 6YB1 and 6YB2. Data supporting the results and conclusions are available within this paper and the Supplementary Information. Additional raw data are available at Figshare, https://doi.org/10.6084/m9.figshare.14406419.

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Acknowledgements

A.J.S. thanks Diamond Light Source for a place on the CCP4 Data Collection and Structure Solution Workshop 2017. E.J.M.L. thanks S. Rao and G. Klesse (University of Oxford) for support with CHAP and F. Marcoline (UCSF) for support with APBSmem. A.J.S. was funded by the Bristol Chemical Synthesis Centre for Doctoral Training funded by the EPSRC (EP/G036764/1). A.N. and K.R.M. were supported by a BBSRC grant to R.L.B., H.B. and D.N.W. (BB/J009784/1). W.M.D., A.N., A.J.S., A.R.T. and D.N.W. were funded by ERC Grants to D.N.W. (340764 and 787173). E.J.M.L. was in the BBSRC/EPSRC-funded Synthetic Biology Research Centre, BrisSynBio (BB/L01386X/1). M.I.W. was funded by the BBSRC (BB/R001790/1). W.F.D. was supported by NIH (R35 GM122603), NSF (1709506) and US Air Force (1709506) grants. H.T.K. was supported by the NIH Ruth L. Kirschstein NRSA Postdoctoral Fellowship (F32 GM125217). M.M. is supported by the Howard Hughes Medical Institute Gilliam Fellowship. D.N.W. held a Royal Society Wolfson Research Merit Award (WM140008). Beamline 8.3.1 at the Advanced Light Source is operated by the University of California Office of the President, Multicampus Research Programs and Initiatives grant MR-15-328599, the National Institutes of Health (R01 GM124149 and P30 GM124169), Plexxikon Inc. and the Integrated Diffraction Analysis Technologies program of the US Department of Energy Office of Biological and Environmental Research. The Advanced Light Source (Berkeley, CA) is a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the US Department of Energy under contract number DE-AC02-05CH11231, Office of Basic Energy Sciences.

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Authors

Contributions

A.J.S., A.N., K.R.M., A.R.T., H.B., R.L.B. and D.N.W. conceived the project and designed the peptides, which A.J.S., A.N. and W.M.D. synthesized. A.J.S., A.N. and K.R.M performed solution-phase biophysics and electrophysiology experiments. A.J.S., H.T.K. and L.L. determined X-ray crystal structures. A.J.S. and A.R.T. did the computational design. E.J.M.L., M.M. and A.J.M. ran and analysed the MD simulations. E.J.M.L. conducted the electrostatic calculations. J.T.S. and M.I.W. conducted and analysed the oSCR. A.J.S. and A.N. contributed equally as first authors to this work. The contributions of H.T.K., E.J.M.L. and J.T.S. were also equal. A.J.S., E.J.M.L. and D.N.W. wrote the manuscript, to which all authors contributed.

Corresponding author

Correspondence to Derek N. Woolfson.

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Supplementary information

Supplementary Information

Computational methods, Supplementary Figs. 1–67, Tables 1–5, Video captions 1–5 and refs. 1–36.

Supplementary Video 1

Molecular dynamics simulation of CC-Type2-TI5.

Supplementary Video 2

Molecular dynamics simulation of CC-Type2-LIS4.

Supplementary Video 3

Molecular dynamics simulation of CC-Type2-TS2.

Supplementary Video 4

oSCR recording of CCTM-VI channels..

Supplementary Video 5

Molecular dynamics simulation of K2-CCTM-VI.

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Scott, A.J., Niitsu, A., Kratochvil, H.T. et al. Constructing ion channels from water-soluble α-helical barrels. Nat. Chem. 13, 643–650 (2021). https://doi.org/10.1038/s41557-021-00688-0

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