A mutational analysis of binding interactions in an antigen-antibody protein-protein complex

William Dall'Acqua, Ellen R. Goldman, Wenhong Lin, Connie Teng, Daisuke Tsuchiya, Hongmin Li, Xavier Ysern, Bradford C. Braden, Yili Li, Sandra J. Smith-Gill, Roy A. Mariuzza

Research output: Contribution to journalArticlepeer-review

143 Scopus citations

Abstract

Alanine scanning mutagenesis, double mutant cycles, and X-ray crystallography were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Twelve out of the 13 nonglycine contact residues on HEL, as determined by the high-resolution crystal structure of the D1.3 - HEL complex, were individually truncated to alanine. Only four positions showed a ΔΔG (ΔG(mutant) - ΔG(wild-type)) of greater than 1.0 kcal/mol, with HEL residue Gln 121 proving the most critical for binding (ΔΔG = 2.9 kcal/mol). These residues form a contiguous patch at the periphery of the epitope recognized by D1.3. To understand how potentially disruptive mutations in the antigen are accommodated in the D1.3- HEL interface, we determined the crystal structure to 1.5 Å, resolution of the complex between D1.3 and HEL mutant Asp18 → Ala. This mutation results in a ΔΔG of only 0.3 kcal/mol, despite the loss of a hydrogen bond and seven van der Waals contacts to the Asp 18 side chain. The crystal structure reveals that three additional water molecules are stably incorporated in the antigen-antibody interface at the site of the mutation. These waters help fill the cavity created by the mutation and form part of a rearranged solvent network linking the two proteins. To further dissect the energetics of specific interactions in the D1.3-HEL interface, double mutant cycles were carried out to measure the coupling of 14 amino acid pairs, 10 of which are in direct contact in the crystal structure. The highest coupling energies, 2.7 and 2.0 kcal/mol, were measured between HEL residue Gln 121 and D1.3 residues V(L)Trp92 and VLTyr32, respectively. The interaction between Gln121 and V(L)Trp92 consists of three van der Waals contacts, while the interaction of Gln121 with V(L)Tyr32 is mediated by a hydrogen bond. Surprisingly, however, most cycles between interface residues in direct contact in the crystal structure showed no significant coupling. In particular, a number of hydrogen-bonded residue pairs were found to make no net contribution to complex stabilization. We attribute these results to accessibility of the mutation sites to water, such that the mutated residues exchange their interaction with each other to interact with water. This implies that the strength of the protein-protein hydrogen bonds in these particular cases is comparable to that of the protein-water hydrogen bonds they replace. Thus, the simple fact that two residues are in direct contact in a protein-protein interface cannot be taken as evidence that there necessarily exists a productive interaction between them. Rather, the majority of such contacts may be energetically neutral, as in the D1.3-HEL complex.

Original languageEnglish (US)
Pages (from-to)7981-7991
Number of pages11
JournalBiochemistry
Volume37
Issue number22
DOIs
StatePublished - May 14 1998
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of 'A mutational analysis of binding interactions in an antigen-antibody protein-protein complex'. Together they form a unique fingerprint.

Cite this