Thermodynamic Analysis of the Binding of 2F5 (Fab and Immunoglobulin G Forms) to Its gp41 Epitope Reveals a Strong Influence of the Immunoglobulin Fc Region on Affinity

Pedro L. Mateo,Sara Crespillo,Salvador Casares,Francisco Conejero-Lara

doi:10.1074/jbc.c113.524439

Abstract

Immunotherapies and vaccines based on the induction of broadly neutralizing monoclonal antibodies (bNAbs) have become outstanding strategies against HIV-1. Diverse bNAbs recognizing different regions of the HIV-1 envelope have been identified and extensively studied. However, there is little information about the thermodynamics of binding of these bNAbs and their epitopes. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We found that these interactions are enthalpically driven and opposed by a negative entropy change. The highest affinity was found for 2F5 IgG for its functional epitope, indicating that additional interactions involving residues flanking the core epitope contribute strongly to higher affinity. In addition, the strong influence of the Fc region on the binding affinity suggests long-range allosteric effects within IgG. Our results provide useful information for developing new therapeutics against HIV-1 and, in a broader scope, contribute to a better understanding of antigen-antibody recognition.

Highlights

Little is known regarding the thermodynamics of binding of the broadly neutralizing anti-HIV-1 mAb 2F5 to its gp41 epitope
Thermodynamics of Binding of 2F5 IgG to Its Epitope—First, a 2F5 IgG solution was titrated with the E7S peptide corresponding to the core epitope of this broadly neutralizing monoclonal antibodies (bNAbs)
A 2F5 IgG solution was titrated with the N16N peptide corresponding to the functional epitope of this bNAb

Summary

Background

Little is known regarding the thermodynamics of binding of the broadly neutralizing anti-HIV-1 mAb 2F5 to its gp epitope. Results: Isothermal titration calorimetry reveals strong differences between IgG and Fab. Conclusion: Residues flanking the core epitope and the immunoglobulin Fc region contribute strongly to affinity by allosteric mechanisms. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb 2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We have characterized thermodynamically the binding of bNAb 2F5 to peptides corresponding to both the core and functional epitopes by isothermal titration calorimetry (ITC). Very few comparative thermodynamic studies of this type by ITC have been reported This information would help to elucidate whether the flexibility of the IgG molecule, mostly around the hinge region between the Fc and Fab regions [26, 27], still allows for an effect on the Ab affinity by allosteric mechanisms. We observed higher binding affinities for the complete IgG than for the Fab fraction, confirming that the immunoglobulin Fc region strongly influences bNAb 2F5 affinity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION