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. 2009 Jun 2;106(22):9057-62.
doi: 10.1073/pnas.0901474106. Epub 2009 May 19.

Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface

Likai Song  1 , Zhen-Yu J Sun, Kate E Coleman, Michael B Zwick, Johannes S Gach, Jia-huai Wang, Ellis L Reinherz, Gerhard Wagner, Mikyung Kim
Affiliations

Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface

Likai Song et al. Proc Natl Acad Sci U S A. .

Abstract

A vaccine capable of stimulating protective antiviral antibody responses is needed to curtail the global AIDS epidemic caused by HIV-1. Although rarely elicited during the course of natural infection or upon conventional vaccination, the membrane-proximal ectodomain region (MPER) of the HIV-1 glycoprotein of M(r) 41,000 (gp41) envelope protein subunit is the target of 3 such human broadly neutralizing antibodies (BNAbs): 4E10, 2F5, and Z13e1. How these BNAbs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but such information might offer important insight into a worldwide health imperative. Here, EPR and NMR techniques were used to define the manner in which these BNAbs differentially recognize viral membrane-encrypted residues configured within the L-shaped helix-hinge-helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identified. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. In contrast, Z13e1 effects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, BNAbs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features important for targeted vaccine design have been revealed, the implications of which extend to BNAb targets on other viral fusion proteins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The structure of the MPER and immersion depth changes induced by 2F5 and Z13e1. (A) Schematic diagram of HIV-1 gp41. FP, fusion peptide region; NHR and CHR, N- and C-terminal α-helices of heptad repeat, respectively; and TM, transmembrane domain. The minimal peptide epitopes of BNAbs are indicated for 2F5 (blue), Z13e1 (green), and 4E10 (magenta). (B) A simple model of env including MPER and TM based on one of the 3-dimensional structural features of HIV-1/SIV-1 trimeric env (–40) and the NMR structure of the HxB2 MPER in a virion mimic membrane (dioleoyl phosphatidylcholine/sphingomyelin/dioleoyl phosphatidylethanolamine/dioleoyl phosphatidylglycerol/cholesterol) surface (blue plane). Residues essential for antibody neutralization are color coded with blue for 2F5, green for Z13e1, and magenta for 4E10. Note that N674 rather than D674 is shown. (C) Immersion depth changes of R1 spin-labeled residues of the MPER upon 2F5 binding. Depth values of MPER R1 residues in the absence and the presence of 2F5 binding are indicated with blue and magenta bars, respectively. Depth values between −5 Å and 0 Å and greater than 0 Å correspond to lipid head group region and acyl chain region, respectively. The precise immersion depths of residues exposed to aqueous phase (depth < −5 Å) cannot be determined experimentally and are thus indicated by the striped bars. The biggest immersion depth changes are boxed in the light blue area. (D) Immersion depth of R1 residues of the MPER in the absence (purple columns) and the presence (yellow columns) of Z13e1.
Fig. 2.
Fig. 2.
Comparison of MPER conformational changes induced by Z13e1 and 4E10. (A) Normalized amide chemical shift changes {calculated as [(ΔHcs)2 + (ΔNcs/5)2]1/2} of Z13e1 Fab-bound (black) and 4E10 Fab-bound (blue) MPERs relative to the unbound peptide in DPC micelles. Indicative chemical shift change (0.1 ppm) is shown with a dotted line. (B) MPER residues involved in Z13e1 (black) and 4E10 (blue) interactions shown by NMR cross-saturation transfer are indicated by relative signal reduction of amide peaks after 250-ms irradiation in methyl region. Cross-saturation transfer efficiencies are higher in Z13e1-bound MPERs because of higher immobility induced by tight Fab binding. The error bars represent statistical errors resulting from uncertainties in peak integration. (C) Comparison of backbone dihedral angles of the MPER in the absence (Middle) and presence of Z13e1 (Top) and 4E10 (Bottom), predicted from chemical shift values of Cα/Cβ. The Φ and Ψ angles are shown in square and triangle symbols, respectively (symbols in white correspond to statistically less reliable predictions). (D) Artistic rendering of MPER orientation changes induced by 4E10, 2F5, and Z13e1. Unbound MPER peptides (yellow tubes) are immersed in the lipid bilayers (light green panel). Red, blue, and green tubes represent the membrane orientation of schematic MPER segments in complex with 4E10, 2F5, and Z13e1 (gray surface area), respectively. N and C termini of MPER peptides are marked by letters N and C, respectively. Several key binding residues' positions are indicated before (yellow) and after (purple) antibody binding. For simplicity, the exact 3-dimensional features of MPER segments and Fabs are not visualized here. (See Fig. S9 for examples of structural details.)
Fig. 3.
Fig. 3.
MPER mobility changes upon Z13e1 binding by NMR and EPR. (A) Ratio of relative NMR intensities (using residue K665 as reference intensity) between Z13e1-bound and unbound MPER peptide (black) and between 4E10-bound and unbound MPER peptide (blue). Residues from S668 to L679 in Z13e1-bound MPER are highly immobile. In contrast, only residues from N671 to I682 in 4E10-bound MPER are highly immobile. (B) EPR spectra of R1 side chains in the MPER. Black traces indicate spectra obtained in the absence of Z13e1. Spectra recorded in the presence of 2-fold molar excess of Z13e1 to peptide are identified by the red traces. Features attributive to highly mobile spectra (W666R1) and highly immobile ones (N671R1 and T676R1) are marked by arrows in white and in red, respectively. All spectra were acquired at 2-mW incident microwave power with a 2-G modulation amplitude and a 100-G scan width. (C) Plot of immobility parameter (the inverse of second moment, 〈H2〉−1) of each of the EPR spectra with bound Z13e1 mAb. Large 〈H2〉−1 value corresponds to highly mobile residues.
Fig. 4.
Fig. 4.
Important contribution of W680 to the 4E10-induced MPER reorientation. (A) Model of MPER segment in complex with 4E10 antibody as viewed from the side. Residues proposed to be involved in 4E10 initial contact (N671 and W680) are in magenta, and reference residues for immersion depth changes are indicated in blue. Of note, the alanine mutations of initial contact residue N671 or core epitope residues W672 and F673 individually abolish 4E10 binding to the membrane-bound MPER by BIAcore analysis (17). (B) BIAcore sensograms of 4E10 binding to MPER variants on liposomes. 4E10 binding to spin-labeled peptides, L669R1/W680A and W678R1/W680A, is compared with that of 4E10 to unlabeled control MPER (HxB2). (C) 4E10-induced EPR spectra changes of spin-labeled side chains with and without W680A mutation. Black and colored traces indicate the spectra in the absence and the presence of 4E10, respectively. The decreased immobility upon 4E10 binding in W680A mutants is indicated by arrows. (D) Immersion depth changes of L669R1 and W678R1 in the control MPER and W680A MPER variant are compared in the absence (black columns) and the presence (gray columns) of 4E10. Depth values corresponding to different membrane regions are plotted as described in Fig. 1.
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