Energetic analysis of an antigen/antibody interface: Alanine scanning mutagenesis and double mutant cycles on the HyHEL-10/lysozyme interaction

Alanine scanning mutagenesis of the HyHEL-10 paratope of the HyHEL-10/HEWL complex demonstrates that the energetically important side chains (hot spots) of both partners are in contact. A plot of Delta Delta G(HyHEL-10_mutant) vs. Delta Delta G(HEWL_mutant) for the five of six interacting side-chain hydrogen bonds is linear (Slope = 1). Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complex when replaced by alanine, but 6 of the 12 HyHEL-10 paratope amino acids do. Double mutant cycle analysis of the single crystallographically identified salt bridge, D32(H)/K97. shows that there is a significant energetic penalty when either partner is replaced with a neutral side-chain amino acid, but the D32(H)N/K97M complex is as stable as the WT. The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing the Delta Delta G values of the Tyr --> Phe vs. the corresponding Tyr --> Ala mutations. The nonpolar contacts in the light chain contribute only about one-half of the total Delta Delta G observed for the Tyr --> Ala mutation, while they are significantly more important in the heavy chain. Replacement of the N31(L)/K96 hydrogen bond with a salt bridge, N31D(L)/K96, destabilizes the complex by 1.4 kcal/mol, The free energy of interaction, Delta Delta G(int), obtained from double mutant cycle analysis showed that Delta Delta G(int) for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL-10 single mutant. Error propagation analysis of double mutant cycles shows that data of atypically high precision an required to use this method meaningfully, except where large Delta Delta G values are analyzed.