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

Abstract

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.

Keywords: cryo-EM; crystallization chaperone; electron microscopy; fiducial marker; membrane protein; transporter.

Figure 1.. The HIV-1 MPER epitope, antibody recognition, and suitability as a structural tag.

a) Structure of MPER epitope (magenta) and bound antibody (10E8v4 pdb:5JNY[36], green) shown relative to the plane of the membrane. Sequence of MPER epitope, and residues critical for binding the antibody 10E8 are shown as sticks, and indicated in the sequence. The asterisk denotes Trp (W) residue from antibody 10E8v4 that interacts with membrane lipids. Approximate position of viral membrane shown with dashed line. b) Structures of Fab fragments bound to the MPER epitope (LN01 pdb:6SNC[27]; VRC42 pdb:6MTQ[24]; DH511 pdb:6P3B[22]; 4E10 pdb:1TZG[42]; Z13E1 pdb:3FN0[59]). Heavy and light chains shown in shades of green, and MPER in magenta. MPER helix is oriented in the same way for each structure, with the approximate position of the viral membrane shown as a yellow slab. Binding affinities for the isolated MPER peptide are shown, as reported in ref.[24, 25, 27, 28, 59]. c) Bioinformatic analysis of MPER fusion and antibody binding to small membrane proteins (see Methods for details). For targets in dataset, pie chart shows the percentage (total number in parentheses) that is (i) compatible with MPER fusion and 10E8v4 or 4E10 antibody fragment binding; (ii) compatible with MPER fusion and antibody binding with <10 clashes between target and antibody fragment; (iii) not compatible with this strategy because the target’s first TM helix is at an oblique angle to the membrane or bound Fab clashes with target. For each category, example models are shown. MPER, antibody fragments, and membrane colored as in panels (a) and (b). Clashes between target and antibody shown in red. For 6EBU, oblique TM1 is shown in chocolate.

Figure 2.. Design of Fluc fluoride channel with N-terminal MPER epitope tag.

a) Construct design. b) Alignment of MPER sequence (pink) to Fluc-Bpe TMH1 sequence (orange), starting at residue 2 (N-terminal methionine not shown). c) Left, gel filtration chromatograms of MPER-Fluc-Bpe. Top trace shows MPER-Fluc-Bpe alone (black trace) and in complex with 10E8v4 (green). Subsequent traces show comparison of gel filtration profiles of MPER-Fluc-Bpe fusion proteins in complex with 10E8v4, with the position of the MPER epitope shifted N-terminally by the indicated number of amino acids. Sequences of register-shifted constructs shown at right. d) Bioinformatic models showing how the Fluc-Bpe/10E8v4 interface would be expected to change for each construct.

Figure 3.. Structural characterization of the Fluc-Ec2/10E8v4 complex.

a) MPER-Fluc-Ec2/10E8v4 crystals b) Left, X-ray crystallographic model of MPER-Fluc-Ec2/10E8v4 complex with 2F_o-F_c electron density map contoured at 1.0 σ. MPER colored magenta, Fluc-Ec2 orange, and 10E8v4 green. Right, 2F_o-F_c composite omit maps for MPER peptide and MPER-Fluc-Ec2 monomer calculated from the MPER-Ec2/10E8v4 data. Maps were calculated omitting 5% of the atoms in the model at a time, contoured at 1.0 σ. c) MPER-Fluc-Ec2/10E8v4 crystal lattice. MPER-Fluc-Ec2 in yellow, and 10E8v4 in green. d) Design of a single-chain variable-domain antibody fragment (scFv) based on 10E8v4. The dashed line indicates the epitope binding interface. e) Gel elution chromatogram of crude periplasmic extract containing scFv f) Left, Gel elution chromatogram of MPER-Fluc-Ec2 incubated with scFv. Middle, MPER-Fluc-Ec2/scFv crystals. Right, Diffraction of MPER-Fluc-Ec2/scFv crystals.

Figure 4.. Negative stain analysis of MPER-Fluc-Ec2 in complex with antibody fragments.

a) Left panel: MPER-Fluc-Ec2/10E8v4 Fab crystal structure shown as surface representation. The dimensions of the expected particles are shown. Middle: Representative negative stain micrograph and 2-D class averages of MPER-Fluc-Ec2/10E8v4 complex. White arrows indicate bound antibody fragments with a characteristic hole between the constant and variable domains. Scale bar, 200Å. Box size, 398Å. Right, 3-D reconstruction of particles. Density envelope has been fitted with the crystal structure from Figure 3. b) Left panel: MPER-Fluc-Ec2/scFv model shown as surface representation. Dimensions of expected particles are shown. Middle: Representative negative stain micrograph and 2-D class averages of MPER-Fluc-Ec2/scFv complex. Scale bar, 300Å. Box size, 398Å. Cartoons are provided as interpretations of the orientation of each class average. Right: 3-D reconstruction of particles. Density envelope has been fitted with bioinformatic model of MPER-Fluc-Ec2/scFv complex.

Figure 5.. Cryo-EM analysis of MPER-Fluc-Bpe in complex with VRC42.

a) Representative negative stain micrograph (left) and 2-D class averages (middle) of MPER-Fluc-Bpe/VRC42 complex (1 dimer: 0.3 Fab molar ratio). Scale bar, 200Å. Box size 320Å. The asterisk denotes the doubly-occupied class, which included 2.5% of total particles. Right, 3-D reconstruction of negative stain particles. Density envelope has been fitted with bioinformatic model based on crystal structures of Fluc-Bpe (pdb:5NKQ, [31]) and VRC42 (pdb:6MTQ [24]). b) Representative cryo-EM micrograph and 2-D class averages of MPER-Fluc-Bpe/VRC42 in vitreous ice. Number of particles present in each class are displayed. Scale bar, 200Å. Box size 328Å. c) 3-D cryo-EM reconstruction of particles. Density envelope has been fitted with model. d) Distribution of viewing angles for particles used in reconstruction.

Figure 6.. 10E8v4 binding to AdiC and GlpF bearing MPER epitope tag.

a) Top: Model of AdiC (dark red) fused with the MPER epitope (magenta) and bound to 10E8v4 Fab (green). Model based on crystal structures of AdiC (pdb: 3NCY)[35] and 10E8v4. b) Left, gel filtration chromatograms of MPER-AdiC alone (gray) and MPER-AdiC/10E8v4 complex (black). Right, SDS-PAGE gel of the indicated fraction with major components labeled. c) Representative negative stain micrograph and 2-D class averages of MPER-AdiC/10E8v4. Cartoons of the AdiC dimer (dark red) and 10E8v4 Fab (green) are shown below each average to assist in interpretation of the orientation. White arrows indicate 10E8v4 Fab. Scale bar, 300Å. Box size, 319Å. d) Model of GlpF tetramer (light blue) fused with the MPER epitope (magenta) and bound to 10E8v4 Fab (green). Model based on crystal structures of GlpF (pdb: 1FX8)[36] and 10E8v4. e) Left, gel filtration chromatograms of the MPER-GlpF alone (gray) and MPER-GlpF/10E8v4 complex (black). Right, SDS-PAGE gel of the indicated fraction with major components labeled. f) Representative negative stain micrograph and 2-D class averages of MPER-GlpF/10E8v4. Cartoon representation of GlpF (light blue) bound to 10E8v4 Fab (green) shown below each class average to assist in interpretation of the orientation. White arrows indicate 10E8v4 Fab. Scale bar, 300Å. Box size, 468Å.