Structural and Thermodynamic Basis of Epitope Binding by Neutralizing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10.
Bottom Line: The conclusions of our structure-function analysis strengthen the idea that to exert effective neutralization, the hydrophobic apex of the solvent-exposed CDR-H3 loop must recognize an antigenic structure more complex than just the linear α-helical epitope and likely constrained by the viral membrane lipids.However, 4E10 (or 4E10-like) antibodies are rarely found in HIV-1-infected individuals or elicited through vaccination.We conclude that the difference between neutralizing and nonneutralizing antibodies of 4E10 is neither structural nor energetic but is related to the capacity to recognize the HIV-1 gp41 epitope inserted in biological membranes.
Affiliation: Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan Biophysics Unit (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, Spain.Show MeSH
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Mentions: Kinetic and thermodynamic parameters governing 4E10ep binding were next examined by SPR as previously described (20, 21). Figure 4 and Fig. S3 in the supplemental material show the sensorgrams corresponding to the binding of the 4E10ep to immobilized WT, WDWD, and ΔLoop Fabs, illustrating the effect of mutations in the apex of the CDR-H3 loop on the association (kon) and dissociation (koff) rate constants. The values of kon and koff for WT Fab at room temperature were 5.2 × 104 M−1 s−1 and 1.1 × 10−3 s−1, respectively, corresponding to a dissociation constant (KD) of 21 nM (Fig. 4A and Table 3). The small value of koff indicates slow dissociation of the peptide from the Fab, a finding consistent with tight binding of the epitope-peptide to the antibody under these experimental conditions. The values of kon, koff, and KD were also determined for the WDWD and ΔLoop Fabs (Fig. 4A and Table 3). Ablation of the hydrophobic CDR-H3 tip did not appreciably affect peptide affinity (KD = 18 nM). Compared to the WT Fab, the slower kon in ΔLoop was compensated by a slower koff, suggesting that the intact loop establishes faster interactions with the peptide, but at the same time its release from the paratope is also accelerated. The slower dissociation rate observed for the ΔLoop Fab could be explained by the stronger H-bond between the GlyH100A(O) of the Fab and Trp680(Nξ) of the peptide (Fig. 2; see also Table S1 in the supplemental material). The double mutation introduced in WDWD Fab decreased the affinity slightly (KD = 41 nM) because of a faster koff with respect to the pWT Fab, which was not compensated with a faster kon (Table 3). Although these differences are arguably small, the data suggest that the more hydrophobic loop of WT Fab facilitated binding of the 4E10ep more effectively than the polar WDWD.
Affiliation: Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan Biophysics Unit (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, Spain.