title: A binding free energy hot spot in the ankyrin repeat protein GABPbeta mediated protein-protein interaction.
abstract: The frequently observed ankyrin repeat motif represents a structural scaffold evolved for mediating protein-protein interactions. As such, these repeats modulate a diverse range of cellular functions. We thermodynamically characterized the heterodimeric GA-binding protein (GABP) alphabeta complex and focused specifically on the interaction mediated by the ankyrin repeat domain of the GABPbeta. Our isothermal titration calorimetric analysis of the interaction between the GABP subunits determined an association constant (K(A)) of 6.0 x 10(8) M(-1) and that the association is favorably driven by a significant change in enthalpy (DeltaH) and a minor change in entropy (-TDeltaS). A total of 16 GABPbeta interface residues were chosen for alanine scanning mutagenesis. The calorimetrically measured differences in the free energy of binding were compared to computationally calculated values resulting in a correlation coefficient r = 0.71. We identified three spatially contiguous hydrophobic and aromatic residues that form a binding free energy hot spot (DeltaDeltaG > 2.0 kcal/mol). One residue provides structural support to the hot spot residues. Three non-hot spot residues are intermediate contributors (DeltaDeltaG approximately 1.0 kcal/mol) and create a canopy-like structure over the hot spot residues to possibly occlude solvent and orientate the subunits. The remaining interface residues are located peripherally and have weak contributions. Finally, our mutational analysis revealed a significant entropy-enthalpy compensation for this interaction.
authors.affiliation: Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06032, USA. desrosiers@student.uchc.edu
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Desrosiers
authors.forename: Zheng-yu
authors.initials: ZY
authors.lastname: Peng
mesh_terms: D000818:Animals; D017089:Ankyrin Repeat; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019277:Entropy; D051268:GA-Binding Protein Transcription Factor / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping; D013816:Thermodynamics
chemical_list: D051268:GA-Binding Protein Transcription Factor; C495658:Gabpb1 protein, mouse