title: Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles.
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.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
authors.forename: E R
authors.initials: ER
authors.lastname: Goldman
authors.forename: W
authors.initials: W
authors.lastname: Dall'Acqua
authors.forename: B C
authors.initials: BC
authors.lastname: Braden
authors.forename: R A
authors.initials: RA
authors.lastname: Mariuzza
mesh_terms: D000936:Antigen-Antibody Complex / Q000737:chemistry*; D015374:Biosensing Techniques; D004527:Egg Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006860:Hydrogen Bonding; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D014461:Ultracentrifugation
chemical_list: D000936:Antigen-Antibody Complex; D004527:Egg Proteins; D007130:Immunoglobulin Idiotypes; D009113:Muramidase