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A designed conformational shift to control protein binding specificity.

Michielssens S, Peters JH, Ban D, Pratihar S, Seeliger D, Sharma M, Giller K, Sabo TM, Becker S, Lee D, Griesinger C, de Groot BL - Angew. Chem. Int. Ed. Engl. (2014)

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.

View Article: PubMed Central - PubMed

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/

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A) Free native ubiquitin has two dominant substates: open and closed. Binding to different binding partners can occur in either the open or the closed substate depending on the binding partner. Ubiquitin is mutated by computational design to stabilize one of the two substates. These mutants are expected to bind selectively to only one class of binding partners. The gray area in the free energy surface indicates the ground state population. B) Computational protocol used to identify mutations shifting the conformational equilibrium and their effect on binding. Fast screening and validation of ubiquitin in the complexes is done using a free energy computational protocol based on the Crooks–Gaussian intersection method. Umbrella sampling simulations were used to compute the free energy profile along the pincer mode. Color code: Blue: stabilized in the open substate or complex binding protein in open substate; red: stabilized in the closed substate or complex binding protein in closed substate; gray: no preference. This color code is maintained in all figures, including the Supporting Information.
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fig01: A) Free native ubiquitin has two dominant substates: open and closed. Binding to different binding partners can occur in either the open or the closed substate depending on the binding partner. Ubiquitin is mutated by computational design to stabilize one of the two substates. These mutants are expected to bind selectively to only one class of binding partners. The gray area in the free energy surface indicates the ground state population. B) Computational protocol used to identify mutations shifting the conformational equilibrium and their effect on binding. Fast screening and validation of ubiquitin in the complexes is done using a free energy computational protocol based on the Crooks–Gaussian intersection method. Umbrella sampling simulations were used to compute the free energy profile along the pincer mode. Color code: Blue: stabilized in the open substate or complex binding protein in open substate; red: stabilized in the closed substate or complex binding protein in closed substate; gray: no preference. This color code is maintained in all figures, including the Supporting Information.

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,[14] 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,[13] which make it impossible to disentangle the effects. In other recent work[15] 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.


A designed conformational shift to control protein binding specificity.

Michielssens S, Peters JH, Ban D, Pratihar S, Seeliger D, Sharma M, Giller K, Sabo TM, Becker S, Lee D, Griesinger C, de Groot BL - Angew. Chem. Int. Ed. Engl. (2014)

A) Free native ubiquitin has two dominant substates: open and closed. Binding to different binding partners can occur in either the open or the closed substate depending on the binding partner. Ubiquitin is mutated by computational design to stabilize one of the two substates. These mutants are expected to bind selectively to only one class of binding partners. The gray area in the free energy surface indicates the ground state population. B) Computational protocol used to identify mutations shifting the conformational equilibrium and their effect on binding. Fast screening and validation of ubiquitin in the complexes is done using a free energy computational protocol based on the Crooks–Gaussian intersection method. Umbrella sampling simulations were used to compute the free energy profile along the pincer mode. Color code: Blue: stabilized in the open substate or complex binding protein in open substate; red: stabilized in the closed substate or complex binding protein in closed substate; gray: no preference. This color code is maintained in all figures, including the Supporting Information.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4497613&req=5

fig01: A) Free native ubiquitin has two dominant substates: open and closed. Binding to different binding partners can occur in either the open or the closed substate depending on the binding partner. Ubiquitin is mutated by computational design to stabilize one of the two substates. These mutants are expected to bind selectively to only one class of binding partners. The gray area in the free energy surface indicates the ground state population. B) Computational protocol used to identify mutations shifting the conformational equilibrium and their effect on binding. Fast screening and validation of ubiquitin in the complexes is done using a free energy computational protocol based on the Crooks–Gaussian intersection method. Umbrella sampling simulations were used to compute the free energy profile along the pincer mode. Color code: Blue: stabilized in the open substate or complex binding protein in open substate; red: stabilized in the closed substate or complex binding protein in closed substate; gray: no preference. This color code is maintained in all figures, including the Supporting Information.
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,[14] 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,[13] which make it impossible to disentangle the effects. In other recent work[15] 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.

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.

View Article: PubMed Central - PubMed

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