Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles

Biochemistry. 1997 Jan 7;36(1):49-56. doi: 10.1021/bi961769k.

Abstract

The idiotope-antiidiotope complex between the anti-hen egg white lysozyme antibody D1.3 and the anti-D1.3 antibody E5.2 provides a useful model for studying protein-protein interactions. A high-resolution crystal structure of the complex is available [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R.J., & Mariuzza, R. A. (1995) Nature 374, 739-742], and both components are easily produced and manipulated in Escherichia coli. We previously analyzed the relative contributions of individual residues of D1.3 to complex stabilization by site-directed mutagenesis [Dall'Acqua, W., Goldman, E. R., Eisenstein, E., & Mariuzza, R. A. (1996) Biochemistry 35, 9667-9676]. In the current work, we introduced single alanine substitutions in 9 out of 21 positions in the combining site of E5.2 involved in contacts with D1.3 and found that 8 of them play a significant role in ligand binding (delta Gmutant-delta Gwild type > 1.5 kcal/mol). Furthermore, energetically important E5.2 and D1.3 residues tend to be juxtaposed in the crystal structure of the complex. In order to further dissect the energetics of specific interactions in the D1.3-E5.2 interface, double mutant cycles were carried out to measure the coupling of 13 amino acid pairs, 9 of which are in direct contact in the crystal structure. The highest coupling energy (4.3 kcal/mol) was measured for a charged-neutral pair which forms a buried hydrogen bond, while side chains which interact through solvated hydrogen bonds have lower coupling energies (1.3-1.7 kcal/mol), irrespective of whether they involve charged-neutral or neutral-neutral pairs. Interaction energies of similar magnitude (1.3-1.6 kcal/mol) were measured for residues forming only van der Waals contacts. Cycles between distant residues not involved in direct contacts in the crystal structure also showed significant coupling (0.5-1.0 kcal/mol). These weak long-range interactions could be due to rearrangements in solvent or protein structure or to secondary interactions involving other residues.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigen-Antibody Complex / chemistry*
  • Biosensing Techniques
  • Egg Proteins / chemistry
  • Egg Proteins / metabolism
  • Escherichia coli / genetics
  • Gene Expression / genetics
  • Hydrogen Bonding
  • Immunoglobulin Idiotypes / immunology*
  • Models, Molecular
  • Muramidase / chemistry*
  • Muramidase / immunology
  • Muramidase / metabolism
  • Mutagenesis, Site-Directed / genetics
  • Mutation / genetics
  • Protein Binding
  • Protein Conformation
  • Ultracentrifugation

Substances

  • Antigen-Antibody Complex
  • Egg Proteins
  • Immunoglobulin Idiotypes
  • Muramidase