A designed conformational shift to control protein binding specificity.
Bottom Line: We demonstrate how in silico designed point mutations within the core of ubiquitin, remote from the binding interface, change the binding specificity by shifting the conformational equilibrium of the ground-state ensemble between open and closed substates that have a similar population in the wild-type protein.Binding affinities determined by NMR titration experiments agree with the predictions, thereby showing that, indeed, a shift in the conformational equilibrium enables us to alter ubiquitin's binding specificity and hence its function.Thus, we present a novel route towards designing specific binding by a conformational shift through exploiting the fact that conformational selection depends on the concentration of binding-competent substates.
Affiliation: Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany) http://www.mpibpc.mpg.de/groups/de_groot/Show MeSH
Mentions: The conformational preference of binding partners for either the open or the closed ubiquitin substate opens the possibility for a novel computational design strategy: rather than optimizing the binding interface, the conformational preference of ubiquitin is shifted to achieve selective binding (Figure 1 A). In native ubiquitin, both substates are similarly populated, allowing complex formation with binding partners that require either the open or the closed substate. Modifying the dynamics such that only one substate is populated should result in selective binding. Our computational protocol (Figure 1 B) serves to design point mutants introducing a conformational shift in the ground-state ensemble. Previous attempts through a combination of computational design and phage display library screening identified potential mutations to achieve a similar effect in ubiquitin.[13,14] However, in these cases at least six combined mutations were required and the mutations were selected based on their affinity for binding partners, not based on the conformational shift in the ground-state ensemble as done here. The results in those previous studies are difficult to interpret in terms of conformational equilibria. In one case, the resulting mutations mainly change the kinetics, which were analyzed using only simple kinetic models, and not the conformational equilibrium. In another case, the mutations combined for a reduction in conformational entropy (by introduction of disulfide bonds), conformational stabilization, and surface mutations, which make it impossible to disentangle the effects. In other recent work several mutants to modulate the ubiquitin system have been identified based on binding affinity. In this functional study the details of the molecular mechanism were secondary and therefore not investigated in detail. In the present study we aim at inducing a conformational shift by selecting mutants solely according to their population along the pincer mode.
Affiliation: Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany) http://www.mpibpc.mpg.de/groups/de_groot/