A stable home for membrane proteins

Membrane proteins play numerous biological roles, such as in signal transduction and viral entry, and are important drug targets. We engineered covalently circularized protein belts that can be used to stabilize patches of phospholipid bilayers, which provide a well-defined, extremely stable and near-native environment for the study of membrane proteins.

HFSP Program Grant holders Andreas Plückthun and Gerhard Wagner and colleagues
authored on Tue, 28 February 2017

Lipid membranes, which surround cells and organelles and thus define biological compartments, contain numerous membrane proteins that perform diverse functions. Studying membrane proteins in their native lipid environment has been challenging and studies are therefore typically performed with membrane proteins extracted from their lipid environment and stabilized by detergent micelles or bicelles. These approaches limit studies of interactions with soluble proteins that may be destabilized by the detergents. More importantly, nanodiscs have the same characteristics as membranes, such as lateral pressure, hydrophobic thickness, and an anisotropic electrostatic field, which affect protein function and are not reproduced by micelles or bicelles. It has previously been recognized that truncated forms of apolipoprotein A1, which are now known as membrane scaffold proteins (MSPs), can be used to surround and stabilize patches of lipid bilayer. However, with the available MSPs, only relatively small nanodiscs could be produced, which limited the number of membrane proteins that could be incorporated.

Figure:  Covalently circularized nanodiscs of different sizes, round (A) or polygonal (B). 1H-15N correlated NMR spectra of neurotensin receptor 1 in complex with neurotensin without (C) and with bound heterotrimeric G protein (D). Cryo-EM images of the interaction between poliovirus and a 50-nm cND decorated with the poliovirus receptor, showing approach, pore formation and ejection of the RNA through the pore (E).

To overcome this problem we used the enzyme sortase A to covalently link the termini of MSPs. The resulting nanodiscs surrounded by covalently circularized MSPs (cNDs) are very stable, can be made with exactly defined diameters, and their shape can be chosen to be round or polygonal.

There are numerous applications of cNDs for the study of membrane proteins by nuclear magnetic resonance (NMR) spectroscopy and single-particle electron microscopy (EM). Tuning of the nanodisc size allowed incorporation of the voltage-dependent anion channel 1 (VDAC1) as a monomer or a dimer in cNDs of 9 and 11 nm diameter, respectively. The neurotensin receptor 1 (NTR1), a G protein-coupled receptor, was embedded in a 9-nm nanodisc without and with bound G protein, allowing NMR studies at temperatures as high as 45°C for weeks without degradation.

The larger cNDs enabled studies of viral entry by single-particle EM. When poliovirus was added to 50-nm cNDs decorated with the poliovirus receptor, we were able to observe the formation of a putative pore and ejection of the viral RNA through the nanodisc. Cryo-EM images promise a first structural characterization of a pore formed by a non-enveloped virus.

Reference

Covalently circularized nanodiscs for studying membrane proteins and viral entry. M.L. Nasr, D. Baptista, M.Strauss, Z.-Y. J. Sun, S. Grigoriu, S. Huser, A. Plückthun, F. Hagn, T. Walz,  J. M. Hogle, G. Wagner . Nature Methods. 2016 Nov 21. doi: 10.1038/nmeth.4079. PMID: 27869813,  PMC5199620

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