Limits...
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.

Show MeSH

Related in: MedlinePlus

Interaction of Fab8066 with 5-helix and design of the corresponding ScFv Sc66.(A) Overall interaction of Fab8066 with 5-helix, (B) detailed view of the interaction of the CDR-H2 loop of Fab8066 (yellow) with the two exposed N-HR helices (green) of 5-helix, and (C) interaction of the CDR-H1 and CDR-H2 loops of Fab8066 (yellow) with two N-HR helices (white) and one C-HR helix (orange) of 5-helix. The addition of a third C-HR helix (transparent orange) to 5-helix to complete the six-helix bundle would result in steric clash with the CDR-H1 and CDR-H2 loops. Color coding in panels A and B is as follows: N-HR and C-HR helices of 5-helix are shown in green and orange, respectively; the CDR heavy and light chain loops of Fab8066 are shown in yellow and white, respectively; the remainder of the heavy and light variable domains are shown in dark red and blue, respectively; the light and heavy constant domains of Fab8066 are shown in pink and light blue, respectively. In panel C, the three N-HR helices are shown in white and residues mapped by alanine scanning mutagenesis of a six-helix bundle construct as the epitope for binding Fab8066 [20], [21], [22], [23], [24], [25], [26], [27], are indicated on one of the N-HR helices (helix Na). Also shown in panel A is the design employed to construct the corresponding ScFv by linking the C-terminus of the light chain variable domain (blue) to the N-terminus of the heavy-chain variable domain (dark red) via a 15-amino acid linker (3×GGGGS). The coordinates are taken from PDB IDs 3MA9 (Fab8066/5-helix complex [35]) and 1SZT (coreS trimer [16]).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4126735&req=5

pone-0104683-g002: Interaction of Fab8066 with 5-helix and design of the corresponding ScFv Sc66.(A) Overall interaction of Fab8066 with 5-helix, (B) detailed view of the interaction of the CDR-H2 loop of Fab8066 (yellow) with the two exposed N-HR helices (green) of 5-helix, and (C) interaction of the CDR-H1 and CDR-H2 loops of Fab8066 (yellow) with two N-HR helices (white) and one C-HR helix (orange) of 5-helix. The addition of a third C-HR helix (transparent orange) to 5-helix to complete the six-helix bundle would result in steric clash with the CDR-H1 and CDR-H2 loops. Color coding in panels A and B is as follows: N-HR and C-HR helices of 5-helix are shown in green and orange, respectively; the CDR heavy and light chain loops of Fab8066 are shown in yellow and white, respectively; the remainder of the heavy and light variable domains are shown in dark red and blue, respectively; the light and heavy constant domains of Fab8066 are shown in pink and light blue, respectively. In panel C, the three N-HR helices are shown in white and residues mapped by alanine scanning mutagenesis of a six-helix bundle construct as the epitope for binding Fab8066 [20], [21], [22], [23], [24], [25], [26], [27], are indicated on one of the N-HR helices (helix Na). Also shown in panel A is the design employed to construct the corresponding ScFv by linking the C-terminus of the light chain variable domain (blue) to the N-terminus of the heavy-chain variable domain (dark red) via a 15-amino acid linker (3×GGGGS). The coordinates are taken from PDB IDs 3MA9 (Fab8066/5-helix complex [35]) and 1SZT (coreS trimer [16]).

Mentions: Crystal structures of Fab8066 and a non-neutralizing Fab (Fab8062) from the same affinity matured series differing in only 4 positions in the CDR-H2 loop, complexed to two mimetics of the pre-haipin intermediate (constructs comprising the N-HR timer surrounded by either two or no C-HR helices resulting in 1∶1 and 1∶3 mimetic:Fab complexes) revealed only subtle structural differences between complexes with the neutralizing and non-neutralizing Fabs [35], [36]. Moreover, despite differences in neutralizing activity of three or more orders of magnitude in Env-pseudotyped virus neutralization assays [22], the binding affinities of the neutralizing and non-neutralizing Fabs to various pre-hairpin intermediate mimetics differ by little more than an order of magnitude [35], [36]. This suggested to us that the ability to bind to the six-helix bundle, in addition to the exposed N-HR trimer of the pre-hairpin intermediate, may be critical to the neutralization activity of this particular series of Fabs. Interestingly, alanine scanning mutagenesis and Western blot analysis showed that this series of Fabs targets a structurally contiguous epitope on the N-HR that is solvent accessible and located in a shallow groove between two C-HR helices in the six-helix bundle (Fig. 1B) [20], [22]. The same residues of the N-HR also comprise part of the binding site in the pre-hairpin intermediate mimetics (Fig. 2A, B) [35], [36]. However, if the Fabs were to bind to the six-helix bundle in the same mode as seen in the crystal structures with the pre-hairpin intermediate mimetics, there would be steric clash and atomic overlap between the CDR-H1 and CDR-H2 loops of the Fab and one of the C-HR helices of the six-helix bundle (Fig. 2C). The simplest hypothesis for these observations is that binding of this series of Fabs to the six-helix bundle involves displacement of one of the C-HR helices.


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)

Interaction of Fab8066 with 5-helix and design of the corresponding ScFv Sc66.(A) Overall interaction of Fab8066 with 5-helix, (B) detailed view of the interaction of the CDR-H2 loop of Fab8066 (yellow) with the two exposed N-HR helices (green) of 5-helix, and (C) interaction of the CDR-H1 and CDR-H2 loops of Fab8066 (yellow) with two N-HR helices (white) and one C-HR helix (orange) of 5-helix. The addition of a third C-HR helix (transparent orange) to 5-helix to complete the six-helix bundle would result in steric clash with the CDR-H1 and CDR-H2 loops. Color coding in panels A and B is as follows: N-HR and C-HR helices of 5-helix are shown in green and orange, respectively; the CDR heavy and light chain loops of Fab8066 are shown in yellow and white, respectively; the remainder of the heavy and light variable domains are shown in dark red and blue, respectively; the light and heavy constant domains of Fab8066 are shown in pink and light blue, respectively. In panel C, the three N-HR helices are shown in white and residues mapped by alanine scanning mutagenesis of a six-helix bundle construct as the epitope for binding Fab8066 [20], [21], [22], [23], [24], [25], [26], [27], are indicated on one of the N-HR helices (helix Na). Also shown in panel A is the design employed to construct the corresponding ScFv by linking the C-terminus of the light chain variable domain (blue) to the N-terminus of the heavy-chain variable domain (dark red) via a 15-amino acid linker (3×GGGGS). The coordinates are taken from PDB IDs 3MA9 (Fab8066/5-helix complex [35]) and 1SZT (coreS trimer [16]).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4126735&req=5

pone-0104683-g002: Interaction of Fab8066 with 5-helix and design of the corresponding ScFv Sc66.(A) Overall interaction of Fab8066 with 5-helix, (B) detailed view of the interaction of the CDR-H2 loop of Fab8066 (yellow) with the two exposed N-HR helices (green) of 5-helix, and (C) interaction of the CDR-H1 and CDR-H2 loops of Fab8066 (yellow) with two N-HR helices (white) and one C-HR helix (orange) of 5-helix. The addition of a third C-HR helix (transparent orange) to 5-helix to complete the six-helix bundle would result in steric clash with the CDR-H1 and CDR-H2 loops. Color coding in panels A and B is as follows: N-HR and C-HR helices of 5-helix are shown in green and orange, respectively; the CDR heavy and light chain loops of Fab8066 are shown in yellow and white, respectively; the remainder of the heavy and light variable domains are shown in dark red and blue, respectively; the light and heavy constant domains of Fab8066 are shown in pink and light blue, respectively. In panel C, the three N-HR helices are shown in white and residues mapped by alanine scanning mutagenesis of a six-helix bundle construct as the epitope for binding Fab8066 [20], [21], [22], [23], [24], [25], [26], [27], are indicated on one of the N-HR helices (helix Na). Also shown in panel A is the design employed to construct the corresponding ScFv by linking the C-terminus of the light chain variable domain (blue) to the N-terminus of the heavy-chain variable domain (dark red) via a 15-amino acid linker (3×GGGGS). The coordinates are taken from PDB IDs 3MA9 (Fab8066/5-helix complex [35]) and 1SZT (coreS trimer [16]).
Mentions: Crystal structures of Fab8066 and a non-neutralizing Fab (Fab8062) from the same affinity matured series differing in only 4 positions in the CDR-H2 loop, complexed to two mimetics of the pre-haipin intermediate (constructs comprising the N-HR timer surrounded by either two or no C-HR helices resulting in 1∶1 and 1∶3 mimetic:Fab complexes) revealed only subtle structural differences between complexes with the neutralizing and non-neutralizing Fabs [35], [36]. Moreover, despite differences in neutralizing activity of three or more orders of magnitude in Env-pseudotyped virus neutralization assays [22], the binding affinities of the neutralizing and non-neutralizing Fabs to various pre-hairpin intermediate mimetics differ by little more than an order of magnitude [35], [36]. This suggested to us that the ability to bind to the six-helix bundle, in addition to the exposed N-HR trimer of the pre-hairpin intermediate, may be critical to the neutralization activity of this particular series of Fabs. Interestingly, alanine scanning mutagenesis and Western blot analysis showed that this series of Fabs targets a structurally contiguous epitope on the N-HR that is solvent accessible and located in a shallow groove between two C-HR helices in the six-helix bundle (Fig. 1B) [20], [22]. The same residues of the N-HR also comprise part of the binding site in the pre-hairpin intermediate mimetics (Fig. 2A, B) [35], [36]. However, if the Fabs were to bind to the six-helix bundle in the same mode as seen in the crystal structures with the pre-hairpin intermediate mimetics, there would be steric clash and atomic overlap between the CDR-H1 and CDR-H2 loops of the Fab and one of the C-HR helices of the six-helix bundle (Fig. 2C). The simplest hypothesis for these observations is that binding of this series of Fabs to the six-helix bundle involves displacement of one of the C-HR helices.

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.

Show MeSH
Related in: MedlinePlus