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Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

Louis JM, Aniana A, Lohith K, Sayer JM, Roche J, Bewley CA, Clore GM - PLoS ONE (2014)

Bottom Line: Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity.The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs.We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

View Article: PubMed Central - PubMed

Affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

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Native-PAGE, SEC-MALS and CD analysis of 6-helix-antibody complexes.(A) Native-PAGE in the presence of increasing molar ratios of Sc66 to 6-helix. 6-helix to Sc66 ratios are shown above the respective lanes and the observed stoichiometry of the complexes (purple, 6-helix; blue, Sc66) and expected molecular weights (kDa) are indicated. (B) SEC-MALS for 6-helix alone (orange) and a 1∶1 mixture of 6-helix with Sc66 (black). Average compositions and masses are indicated next to the peaks. (C) CD spectra of 6-helix alone (black) and a 1∶2 mixture of (6 helix+Sc66) minus Sc66 alone (orange). The CD data indicate that there is no change in helicity of 6-helix upon complexation with Sc66.
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pone-0104683-g005: Native-PAGE, SEC-MALS and CD analysis of 6-helix-antibody complexes.(A) Native-PAGE in the presence of increasing molar ratios of Sc66 to 6-helix. 6-helix to Sc66 ratios are shown above the respective lanes and the observed stoichiometry of the complexes (purple, 6-helix; blue, Sc66) and expected molecular weights (kDa) are indicated. (B) SEC-MALS for 6-helix alone (orange) and a 1∶1 mixture of 6-helix with Sc66 (black). Average compositions and masses are indicated next to the peaks. (C) CD spectra of 6-helix alone (black) and a 1∶2 mixture of (6 helix+Sc66) minus Sc66 alone (orange). The CD data indicate that there is no change in helicity of 6-helix upon complexation with Sc66.

Mentions: The binding of Fab8066 and Sc66 to coreS does not allow one to distinguish whether binding occurs directly to the surface accessible region of the N-HR helices between the surrounding C-HR helices in the six-helix bundle conformation, or whether binding involves displacement (or fraying) of one or more C-HR helices in the six-helix bundle permitting interaction with the resulting fully exposed epitope on the trimeric N-HR coiled coil. To this end we investigated the binding of Fab8066 and Sc66 to coreSP (Fig. 4) and 6-helix (Fig. 5). In the case of coreSP, displacement of the C-HR would result in dissociation of the C-HR helix into free solution (where the free C-HR peptide would adopt a largely random coil structure with a very weak CD helical signature; cf. Fig. 4C and Fig. S4C in File S1), while for 6-helix only a single C-HR could be displaced without unraveling the protein.


Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

Louis JM, Aniana A, Lohith K, Sayer JM, Roche J, Bewley CA, Clore GM - PLoS ONE (2014)

Native-PAGE, SEC-MALS and CD analysis of 6-helix-antibody complexes.(A) Native-PAGE in the presence of increasing molar ratios of Sc66 to 6-helix. 6-helix to Sc66 ratios are shown above the respective lanes and the observed stoichiometry of the complexes (purple, 6-helix; blue, Sc66) and expected molecular weights (kDa) are indicated. (B) SEC-MALS for 6-helix alone (orange) and a 1∶1 mixture of 6-helix with Sc66 (black). Average compositions and masses are indicated next to the peaks. (C) CD spectra of 6-helix alone (black) and a 1∶2 mixture of (6 helix+Sc66) minus Sc66 alone (orange). The CD data indicate that there is no change in helicity of 6-helix upon complexation with Sc66.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126735&req=5

pone-0104683-g005: Native-PAGE, SEC-MALS and CD analysis of 6-helix-antibody complexes.(A) Native-PAGE in the presence of increasing molar ratios of Sc66 to 6-helix. 6-helix to Sc66 ratios are shown above the respective lanes and the observed stoichiometry of the complexes (purple, 6-helix; blue, Sc66) and expected molecular weights (kDa) are indicated. (B) SEC-MALS for 6-helix alone (orange) and a 1∶1 mixture of 6-helix with Sc66 (black). Average compositions and masses are indicated next to the peaks. (C) CD spectra of 6-helix alone (black) and a 1∶2 mixture of (6 helix+Sc66) minus Sc66 alone (orange). The CD data indicate that there is no change in helicity of 6-helix upon complexation with Sc66.
Mentions: The binding of Fab8066 and Sc66 to coreS does not allow one to distinguish whether binding occurs directly to the surface accessible region of the N-HR helices between the surrounding C-HR helices in the six-helix bundle conformation, or whether binding involves displacement (or fraying) of one or more C-HR helices in the six-helix bundle permitting interaction with the resulting fully exposed epitope on the trimeric N-HR coiled coil. To this end we investigated the binding of Fab8066 and Sc66 to coreSP (Fig. 4) and 6-helix (Fig. 5). In the case of coreSP, displacement of the C-HR would result in dissociation of the C-HR helix into free solution (where the free C-HR peptide would adopt a largely random coil structure with a very weak CD helical signature; cf. Fig. 4C and Fig. S4C in File S1), while for 6-helix only a single C-HR could be displaced without unraveling the protein.

Bottom Line: Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity.The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs.We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

View Article: PubMed Central - PubMed

Affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

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