A detailed thermodynamic analysis of ras/effector complex interfaces.
Many cellular functions are based on the interaction and crosstalk of various signaling proteins. Among these, members of the Ras family of small GTP-binding proteins are important for communicating signals into different pathways. In order to answer the question of how binding affinity and specificity is achieved, we analyzed binding energetics on the molecular level, with reference to the available structural data. The interaction of two members of the Ras subfamily with two different effector proteins, namely Raf and RalGDS, were investigated using isothermal titration calorimetry and a fluorescence-based method. Experiments with alanine mutants, located in the complex interfaces, yielded an energy map for the contact areas of the Ras/effector complexes, which could be differentiated into enthalpy and entropy contributions. In addition, by using double mutant cycle analysis, we probed the energetic contribution of selected pairs of amino acid residues. The resulting energy landscapes of the Ras/effector interface areas show a highly different topology when comparing the two effectors, Raf and RalGDS, demonstrating the specificity of the respective interactions. Particularly, we observe a high degree of compensating effects between enthalpy and entropy; differences between these components are much greater than the overall free energy differences. This is observed also when using the software FOLD-X to predict the effect of point mutations on the crystal structures of the different complexes. Prediction of the free energy changes shows a very good correlation with the experimentally observed energies. Furthermore, in line with experimental data, energy decomposition indicates that many different components of large magnitude counteract each other to produce a smaller change in overall free energy, illustrating the importance of long-range electrostatic forces in complex formation.
Kiel
Christina
C
Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Serrano
Luis
L
Herrmann
Christian
C
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D017186:Titrimetry; D020726:ral Guanine Nucleotide Exchange Factor / Q000737:chemistry* / Q000378:metabolism*; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D020726:ral Guanine Nucleotide Exchange Factor; D018631:ras Proteins