Evaluation of
direct and cooperative interactions
in a protein-protein interface
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Protein complexes are stabilized by non-covalent interactions similar to those, which stabilize the folded conformation of a protein (e.g. hydrophobic interactions, hydrogen bonding, electrostatic and van der Waals interactions). The reductionist approach to study such interactions by constructing single mutations is severely limited by the fact that bond properties are related to their specific environment, and are not necessary the sum of the parts.
A more advanced protocol is by constructing double-mutant cycles between pairs of residues. The coupling energy A binding unit consists of a pair of interacting residues plus their immediate neighbors, which may execute secondary effect on this interaction. This multiple mutant was used as reference state to which combinations of side chains were introduced and their interaction energy was determined. Addition of neighboring residues, allows the evaluation of their cooperative effects on the interaction. This approach was initially applied to evaluate the net binding free energy of two buried hydrogen bonds and two salt-bridges, involving Asp 49 on BLIP and four residues on TEM1 (Albeck et al. 2000). Fig. 4 shows one of these interactions (D49-R249) analyzed in details. Using this approach we demonstrated experimentally that the two buried salt-bridges were either neutral or repulsive (Fig 4e), whereas the two hydrogen bonds contributed 0.3 kcal/mol binding free energy each. Conversely, a double-mutant cycle analysis of these interactions in their native environment showed that they all stabilize the complex by 1-1.5 kcal/mol. Examination of the effects of neighboring residues on each of the interactions revealed that the formation of a salt bridge triad, which involves two connected salt bridges, had a strong cooperative effect on stabilizing the complex independent of the presence or absence of additional neighbors (Fig. 4e) (Albeck et al. 2000). This explains the favorable interaction energy measured for the two salt-bridges using the standard double mutant cycle approach (1-1.5 kcal/mol). These results demonstrate the importance of forming net-works of buried salt bridges. Theoretical electrostatic calculations predict the observed mode of cooperativity (Fig. 4f), and suggest that the cooperative networking effect results from the favorable contribution of neighboring residues to this interaction (Albeck et al. 2000). To probe for possible structural rearrangement caused by mutagenesis, all mutant structures were minimized (and found to be similar). In addition, the structure of the BLIP D49A mutant (where all four interactions were lost) in complex with TEM1 was experimentally determined (in collaboration with Strynadka, N. University of British Columbia). No change in structure of this mutant was observed (Fig. 4d). The approach discussed above is now implemented to additional sites within the interface, to analyze cooperativity of other residue types in placed in various environments.
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int, which is obtained by the latter is an experimental measure of the interaction between two residues. However, this energy includes, in addition to the direct interaction energy, environmental effects (due to neighboring residues, cooperativity, water ect.). In order to separate between the direct energetic contribution of a given bond and the environmental effects, the multiple-mutant cycles method was developed. In this study a multiple mutant cycle approach was used to decipher direct and cooperative contributions to binding in the interface between TEM1 and BLIP. This approach involves the mutation to Ala of all side chains within a binding unit.
