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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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Free energy profiles for six different ubiquitin mutants, calculated using umbrella sampling simulations. Mutants preferring the closed substate are shown in red, open substate stabilizing mutants are depicted in blue, those without a preference are shown in gray. The wild-type profile is plotted in black. C refers to the closed substate, O to the open substate.
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fig03: Free energy profiles for six different ubiquitin mutants, calculated using umbrella sampling simulations. Mutants preferring the closed substate are shown in red, open substate stabilizing mutants are depicted in blue, those without a preference are shown in gray. The wild-type profile is plotted in black. C refers to the closed substate, O to the open substate.

Mentions: Of the 126 mutations examined, 15 cases resulted in a significant (>4.184 kJ mol−1 or 1 kcal mol−1) relative stabilization of either the open or closed substate (Figure 2). In addition, the change in folding free energy was estimated[18] to monitor potential destabilization of the protein. As expected, most mutants mildly destabilize wild-type ubiquitin. For the 15 most promising mutations, the destabilization is less than 23.6 kJ mol−1, the folding free energy of the wild-type ubiquitin.[19] An additional computational validation was performed using umbrella sampling simulations (for computational details see the Supporting Information). Although this method requires orders of magnitude more computational time, the methodology provides a complete free energy profile along the pincer mode (see Figure 3, and Figures S8 and S9 in the Supporting Information). Overall, 11 out of the 15 mutations were confirmed by umbrella sampling simulations, and of those 15 mutants I13F, I36F, I36Y,and I36A show significant stabilization of the open substate, whereas L69S and L69T show significant stabilization of the closed substate. A recent study[20] found all six of these mutants to have a negative effect on yeast growth rate, indicating that they significantly interfere with normal ubiquitin function. Also, the L69S mutant was previously described by Fushman and co-workers[19] as a more selective binder than wild-type ubiquitin. We were able to identify the selectivity of this mutant in terms of a shift in the open–closed equilibrium.


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)

Free energy profiles for six different ubiquitin mutants, calculated using umbrella sampling simulations. Mutants preferring the closed substate are shown in red, open substate stabilizing mutants are depicted in blue, those without a preference are shown in gray. The wild-type profile is plotted in black. C refers to the closed substate, O to the open substate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Free energy profiles for six different ubiquitin mutants, calculated using umbrella sampling simulations. Mutants preferring the closed substate are shown in red, open substate stabilizing mutants are depicted in blue, those without a preference are shown in gray. The wild-type profile is plotted in black. C refers to the closed substate, O to the open substate.
Mentions: Of the 126 mutations examined, 15 cases resulted in a significant (>4.184 kJ mol−1 or 1 kcal mol−1) relative stabilization of either the open or closed substate (Figure 2). In addition, the change in folding free energy was estimated[18] to monitor potential destabilization of the protein. As expected, most mutants mildly destabilize wild-type ubiquitin. For the 15 most promising mutations, the destabilization is less than 23.6 kJ mol−1, the folding free energy of the wild-type ubiquitin.[19] An additional computational validation was performed using umbrella sampling simulations (for computational details see the Supporting Information). Although this method requires orders of magnitude more computational time, the methodology provides a complete free energy profile along the pincer mode (see Figure 3, and Figures S8 and S9 in the Supporting Information). Overall, 11 out of the 15 mutations were confirmed by umbrella sampling simulations, and of those 15 mutants I13F, I36F, I36Y,and I36A show significant stabilization of the open substate, whereas L69S and L69T show significant stabilization of the closed substate. A recent study[20] found all six of these mutants to have a negative effect on yeast growth rate, indicating that they significantly interfere with normal ubiquitin function. Also, the L69S mutant was previously described by Fushman and co-workers[19] as a more selective binder than wild-type ubiquitin. We were able to identify the selectivity of this mutant in terms of a shift in the open–closed equilibrium.

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