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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.

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Related in: MedlinePlus

Structure of the llama heavy chain antibody fragment VHH D7.(A) Ribbon representation of D7; the complementarity determining regions (CDR) are highlighted in yellow (CDR1), orange (CDR2) and salmon (CDR3). The first and last residue of each CDR is shown together with the side chain of Trp96 critical for gp120 interaction and neutralization. The dotted line indicates CDR3 residues lacking continuous main chain density for residues Arg100 to Ser100B. (B) A close-up of the CDR interaction network reveals multiple polar interactions between CDR1 and CDR3 as well as CDR2 and CDR3.
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pone-0010482-g001: Structure of the llama heavy chain antibody fragment VHH D7.(A) Ribbon representation of D7; the complementarity determining regions (CDR) are highlighted in yellow (CDR1), orange (CDR2) and salmon (CDR3). The first and last residue of each CDR is shown together with the side chain of Trp96 critical for gp120 interaction and neutralization. The dotted line indicates CDR3 residues lacking continuous main chain density for residues Arg100 to Ser100B. (B) A close-up of the CDR interaction network reveals multiple polar interactions between CDR1 and CDR3 as well as CDR2 and CDR3.

Mentions: The crystal structure of the llama heavy chain antibody fragment VHH D7 was solved by molecular replacement and refined to a resolution of 1.5 Å with an R factor of 16.6% and an Rfree of 19.4% (table 1). D7 folds into a typical immunoglobulin domain closely resembling known llama VHH structures [36] (Figure 1A). It contains two canonical (CDR1 and CDR2) and a long CDR3 typical for llama VHHs [37] with a non-canonical CDR conformation [38]. CDR3 is composed of 18 residues (Lys95 – Tyr102) (Figure 2). The base of CDR3 is well defined and stabilized by multiple main chain and side chain interactions including hydrogen bonds and salt bridges with CDR1 (Ser31-Arg97, Asp33-Lys95, Asp33-Arg97 and Asp33-Ser100F) and CDR2 (Ser52-Asp100C and Thr56-Asp100C) (Figure 1B). The extensive inter CDR stabilization suggests a potentially lower flexibility of the CDRs upon binding to gp120. The CD4 binding site antibody b12 employs only one polar (Ser30-Tyr53) and few hydrophobic inter heavy chain CDR contacts [14], [39]. However, the tip of the D7 CDR3 (Arg100 - Ser100B) is highly mobile evidenced by the lack of continuous main chain density for three residues, including Tyr100A positioned at the apex of CDR3, indicating that their conformational flexibility might be important for gp120 recognition.


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)

Structure of the llama heavy chain antibody fragment VHH D7.(A) Ribbon representation of D7; the complementarity determining regions (CDR) are highlighted in yellow (CDR1), orange (CDR2) and salmon (CDR3). The first and last residue of each CDR is shown together with the side chain of Trp96 critical for gp120 interaction and neutralization. The dotted line indicates CDR3 residues lacking continuous main chain density for residues Arg100 to Ser100B. (B) A close-up of the CDR interaction network reveals multiple polar interactions between CDR1 and CDR3 as well as CDR2 and CDR3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010482-g001: Structure of the llama heavy chain antibody fragment VHH D7.(A) Ribbon representation of D7; the complementarity determining regions (CDR) are highlighted in yellow (CDR1), orange (CDR2) and salmon (CDR3). The first and last residue of each CDR is shown together with the side chain of Trp96 critical for gp120 interaction and neutralization. The dotted line indicates CDR3 residues lacking continuous main chain density for residues Arg100 to Ser100B. (B) A close-up of the CDR interaction network reveals multiple polar interactions between CDR1 and CDR3 as well as CDR2 and CDR3.
Mentions: The crystal structure of the llama heavy chain antibody fragment VHH D7 was solved by molecular replacement and refined to a resolution of 1.5 Å with an R factor of 16.6% and an Rfree of 19.4% (table 1). D7 folds into a typical immunoglobulin domain closely resembling known llama VHH structures [36] (Figure 1A). It contains two canonical (CDR1 and CDR2) and a long CDR3 typical for llama VHHs [37] with a non-canonical CDR conformation [38]. CDR3 is composed of 18 residues (Lys95 – Tyr102) (Figure 2). The base of CDR3 is well defined and stabilized by multiple main chain and side chain interactions including hydrogen bonds and salt bridges with CDR1 (Ser31-Arg97, Asp33-Lys95, Asp33-Arg97 and Asp33-Ser100F) and CDR2 (Ser52-Asp100C and Thr56-Asp100C) (Figure 1B). The extensive inter CDR stabilization suggests a potentially lower flexibility of the CDRs upon binding to gp120. The CD4 binding site antibody b12 employs only one polar (Ser30-Tyr53) and few hydrophobic inter heavy chain CDR contacts [14], [39]. However, the tip of the D7 CDR3 (Arg100 - Ser100B) is highly mobile evidenced by the lack of continuous main chain density for three residues, including Tyr100A positioned at the apex of CDR3, indicating that their conformational flexibility might be important for gp120 recognition.

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