Allostery is a fundamental and ubiquitous regulatory mechanism in many life processes. From both fundamental and biomedical application points of view, it is imperative to develop methodologies that enable systematic identification of “hotspot” residues that dictate the degree of co-operativity between allosteric and active sites. Recent deep mutation analysis of a bacteria transcription factor, TetR(B), observed that allostery hotspots are distributed throughout the protein, in contrast to the popular view that hotspot residues tend to form well-defined pathways for “information transduction”. Remarkably, the loss of allostery due to mutation of hotspot residues could be rescued in multiple ways by additional mutations, suggesting a considerable degree of degeneracy in the allosteric network. The availability of a large amount of mutation data that explicitly probe allostery offers a unique opportunity to comprehensively test algorithms for the prediction of allostery hotspots and design principles of allostery.<br/><br/>To gain molecular level insights into the allosteric network in TetR, we have conducted 50 microseconds unbiased molecular dynamics (MD) simulations for three apo TetR(B) systems, the wildtype (WT), a single mutant with disrupted allostery (dead), and a triple mutant that restored allostery from the dead mutant (rescued); multiple microseconds simulations have also been conducted for the inducer-bound and DNA-bound states for these systems. The long simulations enable us to comprehensively characterize the free energy landscape of TetR(B) and identify the order parameters (OP) that best describe transition among major free energy basins. Computed small angle X-ray scattering profiles for the apo and ligand-bound states agreed well with experimental results, suggesting that the apo state is structurally flexible but well-folded, ruling out a popular model that hypothesized coupled binding/folding equilibrium of the DNA-binding domain. The comparison of free energy landscapes and transition pathways in different TetR(B) variants provide insights into how long-range co-operativity is tuned by shifting the equilibrium properties of key free energy basins. Collectively, these results have laid the ground work for additional computational studies for generating testable predictions of allosteric regulation in TetR(B).

Molecular dynamics analysis of allosteric network in tetracycline repressor

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.