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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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Engineered mimetics of the pre-hairpin intermediate and post-fusion six-helix bundle of HIV-1 gp41.(A) Domain organization of HIV-1 gp41 and sequences of the six-helix bundle (6-helix, coreS and coreSP) and pre-hairpin (5-helix and N35CCG-N13) mimetics. (FP, fusion protein; FPPR, fusion peptide proximal region; N-HR, N-heptad repeat; IL, immune-dominant linker; C-HR, C-heptad repeat; MPER, membrane proximal external region; TM, transmembrane region; CT, intraviral C-terminal domain.) Three N35CCG-N13 chains are linked covalently via intermolecular disulfide bridges (CCG, shown in purple) to form a stable helical trimer [19]. N-HR, C-HR and linker residues are shown in green, orange and black (underlined), respectively. Numbering of N-HR and C-HR regions is according to their location in Env from HIV-1 (strain HXB2). Positions in the helical wheel (blue italic) of N-HR residues that are solvent accessible in the six-helix bundle conformation are indicated. (B) Interactions of Fab8066 with N-HR residues in the context of the six-helix bundle construct coreS mapped by Ala scanning mutagenesis and immunoblotting [22]. The coreS trimer is shown as a surface representation with N-HR and C-HR regions of gp41 in white and light orange, respectively. N-HR surface accessible residues (H564, W571, K574 and Q575) identified as sites of interaction with Fab8066 are shown in distinct colors. (C) Relative migration on SDS-PAGE of the gp41 mimetics used in this study. Molecular weights of constructs and markers (M) are indicated in kDa. N-HR and C-HR denote peptides which assemble to form the six-helix bundle conformation of coreSP. (D) Ribbon representations of the gp41 constructs. 5-helix, CCIZN36 and N35CCG-N13 are pre-hairpin intermediate mimetics with one or more exposed N-HR helices, that are otherwise partially shielded in the six-helix bundle. The three N-HR peptide chains in N35CCG-N13 and CCIZN36 are stabilized as disulfide-linked trimers by fusion with either a 13-residue repeat of the N-HR [19] or an N-terminal isoleucine zipper segment [37], respectively. 6-helix and 5-helix are single chain polypeptides with the N-HR (N) and C-HR (C) regions connected by a six-residue linker in the order N-C-N-C-N-C and N-C-N-C-N, respectively [18]. CoreS and coreSP also form a six-helix bundle but as a trimer consisting of 3 hairpin (N-linker-C) peptides and a hexamer consisting of 3 N-HR and 3 C-HR peptides, respectively (see panel A).
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pone-0104683-g001: Engineered mimetics of the pre-hairpin intermediate and post-fusion six-helix bundle of HIV-1 gp41.(A) Domain organization of HIV-1 gp41 and sequences of the six-helix bundle (6-helix, coreS and coreSP) and pre-hairpin (5-helix and N35CCG-N13) mimetics. (FP, fusion protein; FPPR, fusion peptide proximal region; N-HR, N-heptad repeat; IL, immune-dominant linker; C-HR, C-heptad repeat; MPER, membrane proximal external region; TM, transmembrane region; CT, intraviral C-terminal domain.) Three N35CCG-N13 chains are linked covalently via intermolecular disulfide bridges (CCG, shown in purple) to form a stable helical trimer [19]. N-HR, C-HR and linker residues are shown in green, orange and black (underlined), respectively. Numbering of N-HR and C-HR regions is according to their location in Env from HIV-1 (strain HXB2). Positions in the helical wheel (blue italic) of N-HR residues that are solvent accessible in the six-helix bundle conformation are indicated. (B) Interactions of Fab8066 with N-HR residues in the context of the six-helix bundle construct coreS mapped by Ala scanning mutagenesis and immunoblotting [22]. The coreS trimer is shown as a surface representation with N-HR and C-HR regions of gp41 in white and light orange, respectively. N-HR surface accessible residues (H564, W571, K574 and Q575) identified as sites of interaction with Fab8066 are shown in distinct colors. (C) Relative migration on SDS-PAGE of the gp41 mimetics used in this study. Molecular weights of constructs and markers (M) are indicated in kDa. N-HR and C-HR denote peptides which assemble to form the six-helix bundle conformation of coreSP. (D) Ribbon representations of the gp41 constructs. 5-helix, CCIZN36 and N35CCG-N13 are pre-hairpin intermediate mimetics with one or more exposed N-HR helices, that are otherwise partially shielded in the six-helix bundle. The three N-HR peptide chains in N35CCG-N13 and CCIZN36 are stabilized as disulfide-linked trimers by fusion with either a 13-residue repeat of the N-HR [19] or an N-terminal isoleucine zipper segment [37], respectively. 6-helix and 5-helix are single chain polypeptides with the N-HR (N) and C-HR (C) regions connected by a six-residue linker in the order N-C-N-C-N-C and N-C-N-C-N, respectively [18]. CoreS and coreSP also form a six-helix bundle but as a trimer consisting of 3 hairpin (N-linker-C) peptides and a hexamer consisting of 3 N-HR and 3 C-HR peptides, respectively (see panel A).

Mentions: The surface envelope (Env) glycoproteins of HIV-1, gp120 and gp41, mediate fusion of the viral and cell membranes [1]. The initial events in the fusion process involve the binding of CD4 and the chemokine co-receptor to gp120 triggering a series of conformational changes in both gp120 and gp41 that culminate in fusion of the viral and cell membranes [2], [3], [4], [5], [6], [7]. Early steps in this process, representing a possible “activated” state of gp120/gp41, have recently been visualized by crystallography and cryo-electron microscopy of a soluble cleaved HIV-1 Env trimer [8], [9]. In these Env structures, gp41 is in a pre-fusion state: the trimeric coiled-coil N-heptad repeat (N-HR, residues 542–591) and the C-terminal heptad repeat (C-HR, residues 623–663) do not interact with one another and both structural elements are solvent accessible. This structure approximates the postulated pre-hairpin intermediate in which the viral and cell membranes are bridged via the C- and N-termini of gp41, respectively [4], [10], [11]. The final conformational rearrangement occurs further along the fusion pathway and involves the formation of a six-helix bundle, the so-called fusogenic/post-fusogenic state, in which the N-HR trimeric helical coiled-coil is surrounded by three C-HR helices [12], [13], [14], [15], [16]. The six-helix bundle brings the viral and cell membranes into contact with one another which eventually leads to fusion [11]. Various constructs have been devised to mimic both the pre-hairpin intermediate [17], [18], [19] and six-helix bundle conformations of gp41 (Figs. 1A and D) [12], [16], [18].


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)

Engineered mimetics of the pre-hairpin intermediate and post-fusion six-helix bundle of HIV-1 gp41.(A) Domain organization of HIV-1 gp41 and sequences of the six-helix bundle (6-helix, coreS and coreSP) and pre-hairpin (5-helix and N35CCG-N13) mimetics. (FP, fusion protein; FPPR, fusion peptide proximal region; N-HR, N-heptad repeat; IL, immune-dominant linker; C-HR, C-heptad repeat; MPER, membrane proximal external region; TM, transmembrane region; CT, intraviral C-terminal domain.) Three N35CCG-N13 chains are linked covalently via intermolecular disulfide bridges (CCG, shown in purple) to form a stable helical trimer [19]. N-HR, C-HR and linker residues are shown in green, orange and black (underlined), respectively. Numbering of N-HR and C-HR regions is according to their location in Env from HIV-1 (strain HXB2). Positions in the helical wheel (blue italic) of N-HR residues that are solvent accessible in the six-helix bundle conformation are indicated. (B) Interactions of Fab8066 with N-HR residues in the context of the six-helix bundle construct coreS mapped by Ala scanning mutagenesis and immunoblotting [22]. The coreS trimer is shown as a surface representation with N-HR and C-HR regions of gp41 in white and light orange, respectively. N-HR surface accessible residues (H564, W571, K574 and Q575) identified as sites of interaction with Fab8066 are shown in distinct colors. (C) Relative migration on SDS-PAGE of the gp41 mimetics used in this study. Molecular weights of constructs and markers (M) are indicated in kDa. N-HR and C-HR denote peptides which assemble to form the six-helix bundle conformation of coreSP. (D) Ribbon representations of the gp41 constructs. 5-helix, CCIZN36 and N35CCG-N13 are pre-hairpin intermediate mimetics with one or more exposed N-HR helices, that are otherwise partially shielded in the six-helix bundle. The three N-HR peptide chains in N35CCG-N13 and CCIZN36 are stabilized as disulfide-linked trimers by fusion with either a 13-residue repeat of the N-HR [19] or an N-terminal isoleucine zipper segment [37], respectively. 6-helix and 5-helix are single chain polypeptides with the N-HR (N) and C-HR (C) regions connected by a six-residue linker in the order N-C-N-C-N-C and N-C-N-C-N, respectively [18]. CoreS and coreSP also form a six-helix bundle but as a trimer consisting of 3 hairpin (N-linker-C) peptides and a hexamer consisting of 3 N-HR and 3 C-HR peptides, respectively (see panel A).
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getmorefigures.php?uid=PMC4126735&req=5

pone-0104683-g001: Engineered mimetics of the pre-hairpin intermediate and post-fusion six-helix bundle of HIV-1 gp41.(A) Domain organization of HIV-1 gp41 and sequences of the six-helix bundle (6-helix, coreS and coreSP) and pre-hairpin (5-helix and N35CCG-N13) mimetics. (FP, fusion protein; FPPR, fusion peptide proximal region; N-HR, N-heptad repeat; IL, immune-dominant linker; C-HR, C-heptad repeat; MPER, membrane proximal external region; TM, transmembrane region; CT, intraviral C-terminal domain.) Three N35CCG-N13 chains are linked covalently via intermolecular disulfide bridges (CCG, shown in purple) to form a stable helical trimer [19]. N-HR, C-HR and linker residues are shown in green, orange and black (underlined), respectively. Numbering of N-HR and C-HR regions is according to their location in Env from HIV-1 (strain HXB2). Positions in the helical wheel (blue italic) of N-HR residues that are solvent accessible in the six-helix bundle conformation are indicated. (B) Interactions of Fab8066 with N-HR residues in the context of the six-helix bundle construct coreS mapped by Ala scanning mutagenesis and immunoblotting [22]. The coreS trimer is shown as a surface representation with N-HR and C-HR regions of gp41 in white and light orange, respectively. N-HR surface accessible residues (H564, W571, K574 and Q575) identified as sites of interaction with Fab8066 are shown in distinct colors. (C) Relative migration on SDS-PAGE of the gp41 mimetics used in this study. Molecular weights of constructs and markers (M) are indicated in kDa. N-HR and C-HR denote peptides which assemble to form the six-helix bundle conformation of coreSP. (D) Ribbon representations of the gp41 constructs. 5-helix, CCIZN36 and N35CCG-N13 are pre-hairpin intermediate mimetics with one or more exposed N-HR helices, that are otherwise partially shielded in the six-helix bundle. The three N-HR peptide chains in N35CCG-N13 and CCIZN36 are stabilized as disulfide-linked trimers by fusion with either a 13-residue repeat of the N-HR [19] or an N-terminal isoleucine zipper segment [37], respectively. 6-helix and 5-helix are single chain polypeptides with the N-HR (N) and C-HR (C) regions connected by a six-residue linker in the order N-C-N-C-N-C and N-C-N-C-N, respectively [18]. CoreS and coreSP also form a six-helix bundle but as a trimer consisting of 3 hairpin (N-linker-C) peptides and a hexamer consisting of 3 N-HR and 3 C-HR peptides, respectively (see panel A).
Mentions: The surface envelope (Env) glycoproteins of HIV-1, gp120 and gp41, mediate fusion of the viral and cell membranes [1]. The initial events in the fusion process involve the binding of CD4 and the chemokine co-receptor to gp120 triggering a series of conformational changes in both gp120 and gp41 that culminate in fusion of the viral and cell membranes [2], [3], [4], [5], [6], [7]. Early steps in this process, representing a possible “activated” state of gp120/gp41, have recently been visualized by crystallography and cryo-electron microscopy of a soluble cleaved HIV-1 Env trimer [8], [9]. In these Env structures, gp41 is in a pre-fusion state: the trimeric coiled-coil N-heptad repeat (N-HR, residues 542–591) and the C-terminal heptad repeat (C-HR, residues 623–663) do not interact with one another and both structural elements are solvent accessible. This structure approximates the postulated pre-hairpin intermediate in which the viral and cell membranes are bridged via the C- and N-termini of gp41, respectively [4], [10], [11]. The final conformational rearrangement occurs further along the fusion pathway and involves the formation of a six-helix bundle, the so-called fusogenic/post-fusogenic state, in which the N-HR trimeric helical coiled-coil is surrounded by three C-HR helices [12], [13], [14], [15], [16]. The six-helix bundle brings the viral and cell membranes into contact with one another which eventually leads to fusion [11]. Various constructs have been devised to mimic both the pre-hairpin intermediate [17], [18], [19] and six-helix bundle conformations of gp41 (Figs. 1A and D) [12], [16], [18].

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