Reply to “The Broadly Neutralizing, Anti-HIV Antibody 4E10: an Open and Shut Case?”

Abstract

W e thank Drs. Strong and Finton for their interest in our work involving the broadly neutralizing anti-HIV-1 antibody 4E10 (1). In that study, we determined the crystal structure of the unbound form of the Fab region of 4E10, as well as that of nonneutralizing mutants with the peptide epitope bound (1). In particular, the conformation of the critical CDR-H3 loop in the crystal structure of the unbound Fab differed greatly from that previously observed for the Fv version of the same antibody (2). Collectively, our data support the existence of a preformed crevice for binding of the helical membrane-proximal external region (MPER) epitope.In the accompanying letter (3), Strong and Finton concur with the main tenet of our article, i.e., that 4E10 must "recognize an antigenic structure more complex than just the linear ␣-helical epitope." However, in the same letter these authors disagree with our interpretation of the structural data corresponding to the new unbound form of the Fab fragment and with our criticism of the structure of the unbound form of the Fv version (2). Strong and Finton argue that the low pH of the crystallization solution and the use of the shorter Fv construct are not compelling arguments to explain the conformational discrepancies with respect to the Fab. They argue that the Fv construct of 4E10 "retains the structure, binding, and neutralization properties of the Fab" (4). In addition, they explain that the thermodynamic signature of the binding (low entropic component) would not be consistent with an order-disorder transition during binding of the epitope peptide in solution. Moreover, based on a qualitative crystal contact analysis, they argue that our structure of the Fab in the unbound state does not reflect the "fully-free state of 4E10, but, unluckily, recapitulates the ligand-bound state, resolving the potential contradiction." The closed conformation observed in the Fv construct may generate a lipid-binding site representing an alternative conformation to the epitope-binding structure, implying that a conformational transition must occur between both forms for effective engagement of the gp41 antigen (2, 4).We find the arguments expressed in the letter by Strong and Finton reasonable, but only to some extent. For example, we acknowledge that the discrepancy between the crystal structures of the unbound form of the Fab and Fv constructs does not by itself constitute sufficient evidence to completely rule out the existence of large conformational changes in the CDR-H3 loop (2). In addition, neither the crystal structure of the unbound Fv nor the "shut-open" mechanism precludes the existence of a distinct unbound form of the antibody competent in binding to the helical epitope. In the framework of the model described in Strong and Finton's letter, the structure described in our paper would correspond to the "open-unbound" antibody.Nevertheless, a fair critical assessment of the unbound structures should take into account the entire data set available in our study (1) and the structures...

Functional Contacts between MPER and the Anti-HIV-1 Broadly Neutralizing Antibody 4E10 Extend into the Core of the Membrane

“…Specially, it is unclear how these residues contribute to the recognition of the epitope in the context of the viral membrane where MPER resides. These questions are more relevant in view that the hydrophobic residues of the apex region of the CDRH3 remain in a conformation exposed towards the solvent, and not oriented towards the bound peptide as it could be expected from their critical role in the virus neutralization by the antibody [7–12]. In contrast, the functionally similar bNAb 10E8 positions the apex of the CDRH3 loop in close proximity to the MPER peptide [13,14] thus rationalizing its neutralization potency in terms of binding affinity to the epitope in membranes.…”

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

“…Strikingly, the structure of the paratope with lipid bound would differ from that of the paratope with peptide epitope bound (10). Nonethe-less, some of these concepts have been challenged by the recent resolution of crystal structures of the unbound Fab form (18,19) and Fab-lipid complexes (12,20).…”