Limits...
Crystal structure of the neutralizing Llama V(HH) D7 and its mode of HIV-1 gp120 interaction.

Hinz A, Lutje Hulsik D, Forsman A, Koh WW, Belrhali H, Gorlani A, de Haard H, Weiss RA, Verrips T, Weissenhorn W - PLoS ONE (2010)

Bottom Line: Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction.Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance.Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site.

View Article: PubMed Central - PubMed

Affiliation: Unit of Virus Host Cell Interactions (UVHCI), UMI 3265, Université Joseph Fourier-EMBL-CNRS, Grenoble, France.

ABSTRACT
HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment V(HH) D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 V(HH) at 1.5 A resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site.

Show MeSH

Related in: MedlinePlus

Surface representation of the CDRs.(A) Surface representation of D7 revealing the gp120 docking interface based on gp120 binding and HIV-1 neutralization results. The CDRs are coloured as in figure 1. CDR3 residues affecting gp120 interaction and HIV-1 IIIB neutralization are indicated in white and residue differences between D7 and A12 are labelled in black. (B) Electrostatic potential map of the surface generated by the CDRs.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2864739&req=5

pone-0010482-g004: Surface representation of the CDRs.(A) Surface representation of D7 revealing the gp120 docking interface based on gp120 binding and HIV-1 neutralization results. The CDRs are coloured as in figure 1. CDR3 residues affecting gp120 interaction and HIV-1 IIIB neutralization are indicated in white and residue differences between D7 and A12 are labelled in black. (B) Electrostatic potential map of the surface generated by the CDRs.

Mentions: In order to confirm the role of CDR3 and the correlation of gp120 interaction and neutralization, wild type D7 and mutants were tested in a TZM-b1 neutralization assay against HIV-1 IIIB. Comparison of the IC50 values reinforces the importance of CDR3. Mutations that lead to decreased gp120 interaction, namely Ala mutations of Lys95 and Trp96, showed a 100-fold increase in IC50, and Leu99 revealed a 10-fold increase in IC50 as compared to D7 wild type (table 4). In contrast, mutation of Tyr100A and Asp100C show a modest decrease in the IC50 (table 4) consistent with a slightly increased affinity for gp120 (table 3). The largest positive effect was observed for the double mutant Asn101Tyr102, which shows a ∼10-fold increase in affinity (table 3) and a reduction of the IC50 value by a factor of 5 (table 4). This underlines the important role of CDR3 (Figure 4A) for neutralization of HIV-1. Since the CD4 binding site on gp120 is negatively charged [13] CDR3 could provide some complementary basic charge for interaction (Figure 4B). Together these findings indicate that the differences related to CDR3 constitute an important factor accounting for the broader neutralization profile of A12 compared to D7 [35] since both CDR1 and CDR2 are almost identical (Figure 2). It is thus possible that C8 employs different structural principles for gp120 interaction and neutralization [35].


Crystal structure of the neutralizing Llama V(HH) D7 and its mode of HIV-1 gp120 interaction.

Hinz A, Lutje Hulsik D, Forsman A, Koh WW, Belrhali H, Gorlani A, de Haard H, Weiss RA, Verrips T, Weissenhorn W - PLoS ONE (2010)

Surface representation of the CDRs.(A) Surface representation of D7 revealing the gp120 docking interface based on gp120 binding and HIV-1 neutralization results. The CDRs are coloured as in figure 1. CDR3 residues affecting gp120 interaction and HIV-1 IIIB neutralization are indicated in white and residue differences between D7 and A12 are labelled in black. (B) Electrostatic potential map of the surface generated by the CDRs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864739&req=5

pone-0010482-g004: Surface representation of the CDRs.(A) Surface representation of D7 revealing the gp120 docking interface based on gp120 binding and HIV-1 neutralization results. The CDRs are coloured as in figure 1. CDR3 residues affecting gp120 interaction and HIV-1 IIIB neutralization are indicated in white and residue differences between D7 and A12 are labelled in black. (B) Electrostatic potential map of the surface generated by the CDRs.
Mentions: In order to confirm the role of CDR3 and the correlation of gp120 interaction and neutralization, wild type D7 and mutants were tested in a TZM-b1 neutralization assay against HIV-1 IIIB. Comparison of the IC50 values reinforces the importance of CDR3. Mutations that lead to decreased gp120 interaction, namely Ala mutations of Lys95 and Trp96, showed a 100-fold increase in IC50, and Leu99 revealed a 10-fold increase in IC50 as compared to D7 wild type (table 4). In contrast, mutation of Tyr100A and Asp100C show a modest decrease in the IC50 (table 4) consistent with a slightly increased affinity for gp120 (table 3). The largest positive effect was observed for the double mutant Asn101Tyr102, which shows a ∼10-fold increase in affinity (table 3) and a reduction of the IC50 value by a factor of 5 (table 4). This underlines the important role of CDR3 (Figure 4A) for neutralization of HIV-1. Since the CD4 binding site on gp120 is negatively charged [13] CDR3 could provide some complementary basic charge for interaction (Figure 4B). Together these findings indicate that the differences related to CDR3 constitute an important factor accounting for the broader neutralization profile of A12 compared to D7 [35] since both CDR1 and CDR2 are almost identical (Figure 2). It is thus possible that C8 employs different structural principles for gp120 interaction and neutralization [35].

Bottom Line: Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction.Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance.Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site.

View Article: PubMed Central - PubMed

Affiliation: Unit of Virus Host Cell Interactions (UVHCI), UMI 3265, Université Joseph Fourier-EMBL-CNRS, Grenoble, France.

ABSTRACT
HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment V(HH) D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 V(HH) at 1.5 A resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site.

Show MeSH
Related in: MedlinePlus