Modulation of Allostery with Multiple Mechanisms by Hotspot Mutations in TetR.

Modulating allosteric coupling offers unique opportunities for biomedical applications. Such efforts can benefit from efficient prediction and evaluation of allostery hotspot residues that dictate the degree of co-operativity between distant sites. We demonstrate that effects of allostery hotspot mutations can be evaluated qualitatively and semi-quantitatively by molecular dynamics simulations in a bacterial tetracycline repressor (TetR). The simulations recapitulate the effects of these mutations on abolishing the induction function of TetR and provide a rationale for the different degrees of rescuability observed to restore allosteric coupling of the hotspot mutations. We demonstrate that the same non-inducible phenotype could be the result of perturbations in distinct structural and energetic properties of TetR. Our work underscore the value of explicitly computing the functional free energy landscapes to effectively evaluate and rank hotspot mutations despite the prevalence of compensatory interactions, and therefore provide quantitative guidance to allostery modulation for therapeutic and engineering applications.