Peripheral Membrane Interactions Boost the Engagement by an Anti-HIV-1 Broadly Neutralizing Antibody

Abstract

The 4E10 antibody displays an extreme breadth of HIV-1 neutralization and therefore constitutes a suitable model system for structure-guided vaccine design and immunotherapeutics against AIDS. In this regard, the relevance of autoreactivity with membrane lipids for the biological function of this antibody is still a subject of controversy. To address this dispute, herein we have compared the membrane partitioning ability of the 4E10 antibody and several of its variants, which were mutated at the region of the paratope surface in contact with the membrane interface. We first employed a physical separation approach (vesicle flotation) and subsequently carried out quantitative fluorescence measurements in an intact system (spectroscopic titration), using 4E10 Fab labeled with a polarity-sensitive fluorescent probe. Moreover, recognition of epitope peptide in membrane was demonstrated by photo-cross-linking assays using a Fab that incorporated the genetically encoded unnatural amino acid -benzoylphenylalanine. The experimental data ruled out that the proposed stereospecific recognition of viral lipids was necessary for the function of the antibody. In contrast, our data suggest that nonspecific electrostatic interactions between basic residues of 4E10 and acidic phospholipids in the membranes contribute to the observed biological function. Moreover, the energetics of membrane partitioning indicated that 4E10 behaves as a peripheral membrane protein, tightening the binding to the ligand epitope inserted in the viral membrane. The implications of these findings for the natural production and biological function of this antibody are discussed.

Surface-Matrix Screening Identifies Semi-specific Interactions that Improve Potency of a Near Pan-reactive HIV-1-Neutralizing Antibody

“…Diverse studies ranging from electron paramagnetic resonance (EPR) analysis (Song et al, 2009; Sun et al, 2008) to mutational and partitioning analyses (Ofek et al, 2010; Rujas et al, 2017), to crystallographic analysis of Fab in complex with phospholipid headgroups (Irimia et al, 2016, 2017) indicate antibodies 10E8 and 4E10 to co-recognize membrane and MPER peptide. In particular, the orientation suggested by the structure of 10E8 with 1,2 dihexanoyl- sn -glycerol-3-phospho-(1’- rac -glycerol) and scaffolded MPER (Irimia et al, 2017) would position the indole ring of Trp100c HC to extend into the viral lipid membrane (Figure 4A).…”

Functional Optimization of Broadly Neutralizing HIV-1 Antibody 10E8 by Promotion of Membrane Interactions

“…The ability to access the helical MPER epitope at the viral membrane interface thus appears to support the neutralizing activity of the most effective anti-MPER antibodies (3,22). Structural elements of the antibody sustain effective interactions with the lipid bilayer surrounding the viral particle: (i) a long heavy chain complementarity-determining region 3 (CDRH3) loop decorated at the apex with hydrophobic-at-interface aromatic residues critical for function (3,8,23,24) and (ii) a flat surface at the paratope that establishes favorable interactions at the viral Env-membrane interface (4,22,25).…”

“…Recent studies suggested that the association of anti-MPER antibodies with membranes might be driven by electrostatic interactions between basic residues on the surface of the paratope and anionic phospholipids (4,22,25). Despite the inability of 10E8 to partition spontaneously into lipid bilayers (25), cryo-electron microscopy (EM) and X-ray crystallography data suggest that upon engagement with the antigen, a surface patch of its paratope may establish favorable contacts with the membrane-Env interface (2-4).…”

“…The ability to access the helical MPER epitope at the viral membrane interface thus appears to support the neutralizing activity of the most effective anti-MPER antibodies (3,22). Structural elements of the antibody sustain effective interactions with the lipid bilayer surrounding the viral particle: (i) a long heavy chain complementarity-determining region 3 (CDRH3) loop decorated at the apex with hydrophobic-at-interface aromatic residues critical for function (3,8,23,24) and (ii) a flat surface at the paratope that establishes favorable interactions at the viral Env-membrane interface (4,22,25).Recent studies suggested that the association of anti-MPER antibodies with membranes might be driven by electrostatic interactions between basic residues on the surface of the paratope and anionic phospholipids (4,22,25). Despite the inability of 10E8 to partition spontaneously into lipid bilayers (25), cryo-electron microscopy (EM) and X-ray crystallography data suggest that upon engagement with the antigen, a surface patch of its paratope may establish favorable contacts with the membrane-Env interface (2-4).…”

The Binding of Aβ42 Peptide Monomers to Sphingomyelin/Cholesterol/Ganglioside Bilayers Assayed by Density Gradient Ultracentrifugation

“…To our knowledge Aβ42 binding to bilayers after separation of the free and lipid-bound forms of the peptide by density gradient centrifugation have rarely been performed. Vesicle-bound peptide floats in the density gradient whereas the non-bound protein/peptide, after aggregation, sediments [24,25]. The centrifugation method is quite unique in providing direct, quantitative equilibrium measurements of binding, in the absence of added probes or chemical modification of the ligand.…”