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Structure determination protocol for transmembrane domain oligomers

Nature Protocols volume 14, pages 2483–2520 (2019)Cite this article

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Abstract

The transmembrane (TM) anchors of cell surface proteins have been one of the ‘blind spots’ in structural biology because they are generally very hydrophobic, sometimes dynamic, and thus difficult targets for structural characterization. A plethora of examples show these membrane anchors are not merely anchors but can multimerize specifically to activate signaling receptors on the cell surface or to stabilize envelope proteins in viruses. Through a series of studies of the TM domains (TMDs) of immune receptors and viral membrane proteins, we have established a robust protocol for determining atomic-resolution structures of TM oligomers by NMR in bicelles that closely mimic a lipid bilayer. Our protocol overcomes hurdles typically encountered by structural biology techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM) when studying small TMDs. Here, we provide the details of the protocol, covering five major technical aspects: (i) a general method for producing isotopically labeled TM or membrane-proximal (MP) protein fragments that involves expression of the protein (which is fused to TrpLE) into inclusion bodies and releasing the target protein by cyanogen bromide (CNBr) cleavage; (ii) determination of the oligomeric state of TMDs in bicelles; (iii) detection of intermolecular contacts using nuclear Overhauser effect (NOE) experiments; (iv) structure determination; and (v) paramagnetic probe titration (PPT) to characterize the membrane partition of the TM oligomers. This protocol is broadly applicable for filling structural gaps of many type I/II membrane proteins. The procedures may take 3–6 months to complete, depending on the complexity and stability of the protein sample.

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Fig. 1: Protocol overview.
Fig. 2: Schematic of the pMM-LR6 vector.
Fig. 3: Expression, purification, and bicelle reconstitution of TMDs.
Fig. 4: Pulse sequence of the 3D JCH-modulated NOE experiment.
Fig. 5: Characterization of the oligomeric state of TMDs in bicelles.
Fig. 6: Detection of interchain NOEs.
Fig. 7: Sample mixing profiles for TMDs.
Fig. 8: Structure determination of TMDs.
Fig. 9: The PPT method for determination of the protein membrane partition.
Fig. 10: Analysis of the protein TM partition.

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Code availability

The code and instructions for the ExSSO program are freely accessible from the website: http://www.csbio.sjtu.edu.cn/bioinf/ExSSO/. The software is also provided as Supplementary Software 1 and 2.

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Acknowledgements

This work was supported by US National Institutes of Health grants GM116898 and AI127193 to J.J.C.

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Author notes

  1. These authors contributed equally: Qingshan Fu, Alessandro Piai, Wen Chen.

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Qingshan Fu, Alessandro Piai, Wen Chen & James J. Chou

  2. Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

    Ke Xia

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Contributions

Q.F., A.P., W.C., and J.J.C. conceived the study. K.X. performed the mass spectrometry analysis. Q.F., A.P., W.C., and J.J.C. wrote the manuscript.

Corresponding author

Correspondence to James J. Chou.

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Related links

Key references using this protocol

Fu, Q. et al. Mol. Cell 61, 602–613 (2016): https://doi.org/10.1016/j.molcel.2016.01.009

Piai, A., Devi, J., Fu, Q., & Chou, J. J. J. Am. Chem. Soc. 139, 18432–18435 (2017): https://doi.org/10.1021/jacs.7b09352

Chen, W. et al. Structure 26, 627–634.e4 (2018): https://doi.org/10.1016/j.str.2018.02.011

Fu, Q. et al. Proc. Natl. Acad. Sci. USA 115, E8892–E8899 (2018): https://doi.org/10.1073/pnas.1807259115

Supplementary information

Supplementary Software 1

ExSSO software for Linux.

Reporting Summary

Supplementary Software 2

ExSSO software for Mac OS.

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Fu, Q., Piai, A., Chen, W. et al. Structure determination protocol for transmembrane domain oligomers. Nat Protoc 14, 2483–2520 (2019). https://doi.org/10.1038/s41596-019-0188-9

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