Abstract
The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER). The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab). However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation.
Highlights
The membrane proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) envelope transmembrane glycoprotein, gp41, is the target of the two broadly neutralizing monoclonal antibodies (Mabs) 2F5 and 4E10 [1,2] (Figure 1A)
The envelope glycoproteins must possess an inherent capacity for conformational change, and consistent with this requirement, the MPER region is well recognized for its conformational diversity
We found a direct correlation between the antibody affinity constant for the MPER peptide and the inhibitory concentration (IC50) of HIV-1 neutralization that suggests a new property of the 2F5 CDRH3 loop that may be additional to and independent of its suggested membrane interaction
Summary
The membrane proximal external region (MPER) of the HIV-1 envelope transmembrane glycoprotein, gp, is the target of the two broadly neutralizing monoclonal antibodies (Mabs) 2F5 and 4E10 [1,2] (Figure 1A). Cellular receptors interacting with gp120 induce conformational changes in both gp120 and gp41 These conformation changes lead to the insertion of the gp Nterminal fusion peptide into the target cell membrane, eventually permitting fusion of the two membranes as gp collapses into the six-helix bundle state, mediating entry of HIV genetic information into the target cell. To mediate this process, the envelope glycoproteins must possess an inherent capacity for conformational change, and consistent with this requirement, the MPER region is well recognized for its conformational diversity. The 2F5 Mab affinity for its epitope is enhanced when
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