N-terminal Transmembrane-Helix Epitope Tag for X-ray Crystallography and Electron Microscopy of Small Membrane Proteins.

Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common strategy to augment such proteins; however, generating antibody fragments that specifically bind a target protein is not trivial. Here we identify a helical epitope, from the membrane-proximal external region (MPER) of the gp41-transmembrane subunit of the HIV envelope protein, that is recognized by several well-characterized antibodies and that can be fused as a contiguous extension of the N-terminal transmembrane helix of a broad range of membrane proteins. To analyze whether this MPER-epitope tag might aid structural studies of small membrane proteins, we determined an X-ray crystal structure of a membrane protein target that does not crystallize without the aid of crystallization chaperones, the Fluc fluoride channel, fused to the MPER epitope and in complex with antibody. We also demonstrate the utility of this approach for single particle electron microscopy with Fluc and two additional small membrane proteins that represent different membrane protein folds, AdiC and GlpF. These studies show that the MPER epitope provides a structurally defined, rigid docking site for antibody fragments that is transferable among diverse membrane proteins and can be engineered without prior structural information. Antibodies that bind to the MPER epitope serve as effective crystallization chaperones and electron microscopy fiducial markers, enabling structural studies of challenging small membrane proteins.
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: B.C.M. and R.B.S. are inventors on a pending patent application filed by the University of Michigan on the use of the MPER epitope for structural biology applications. The other authors declare no competing interests. Materials availability The 10E8v4 scFv plasmid generated in this study has been deposited to AddGene. Plasmids with MPER-tagged AdiC, Fluc-Bpe, Fluc-Ec2, and GlpF genes are available upon request. Data availability Atomic coordinates for the crystal structure of MPER-Fluc-Ec2/10E8v4 have been deposited in the Protein Data Bank under accession number 6X58. The cryo-EM map of MPER-Fluc-Bpe/VRC42 has been deposited in the Electron Microscopy Database under accession number EMD-23247. Models generated for the bioinformatic analysis in Figure 1 is available for download from the Deep Blue Data repository hosted by the University of Michigan, with unique identifier https://doi.org/10.7302/7ym7-gp78. No custom code was used.
(Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)