Thermodynamic Signatures of the Antigen Binding Site of mAb 447–52D Targeting the Third Variable Region of HIV‑1 gp120

Abstract

The third variable region (V3) of HIV-1 gp120 plays a key role in viral entry into host cells; thus, it is a potential target for vaccine design. Human monoclonal antibody (mAb) 447–52D is one of the most broadly and potently neutralizing anti-V3 mAbs. We further characterized the 447–52D epitope by determining a high-resolution crystal structure of the Fab fragment in complex with a cyclic V3 and interrogated the antigen–antibody interaction by a combination of site-specific mutagenesis, isothermal titration calorimetry (ITC) and neutralization assays. We found that 447–52D’s neutralization capability is correlated with its binding affinity and at 25 °C the Gibbs free binding energy is composed of a large enthalpic component and a small favorable entropic component. The large enthalpic contribution is due to (i) an extensive hydrogen bond network, (ii) a π–cation sandwiching the V3 crown apex residue Arg³¹⁵, and (iii) a salt bridge between the 447–52D heavy chain residue Asp^H95 and Arg³¹⁵. Arg³¹⁵ is often harbored by clade B viruses; thus, our data explained why 447–52D preferentially neutralizes clade B viruses. Interrogation of the thermodynamic signatures of residues at the antigen binding interface gives key insights into their contributions in the antigen–antibody interaction