Allostery is a fundamental and ubiquitous regulatory mechanism in many life processes. It is imperative to identify allostery hotspots that regulate the degree of cooperativity between allosteric and active sites in a comprehensive and unbiased fashion. A recent deep mutational scanning study of the tetracycline repressor (TetR) revealed an unexpectedly broad distribution of hotspots throughout the protein structure. The loss of allostery due to mutation of hotspot residues could also be rescued in multiple ways by additional mutations, suggesting a considerable degree of degeneracy in the allosteric network.<br/><br/>Using extensive molecular dynamics simulations (up to 50 μs) and free energy computations, we establish the molecular and energetic basis for the strong anti-cooperativity between the ligand and DNA binding sites in the wild type TetR. The computed free energy landscapes in different ligation states illustrate that allostery in TetR is well described by a conformational selection model. By examining a range of structural and dynamic properties of residues at both local and global scales, we observe that various analyses capture different subsets of experimentally identified hotspots, suggesting that these residues modulate allostery in distinct ways. These results motivate the development of a thermodynamic model that qualitatively explains the broad distribution of hotspot residues and their distinct features, as well as the mutation effects in molecular dynamics simulations. Two types of hotspots may contribute by either mediating inter-domain communications or intra-domain energetics. A single mutant with disrupted allostery (dead) and a triple mutant that restored allostery from the dead mutant (rescued) are studied to further understand the mutation effects on different ligation states relative to the wild type in terms of both structural changes and energetics. Extracting the thermodynamic parameters based on mutant studies would help build predictive computational models to explore higher order mutations to design novel phenotypes in allosteric systems. The multi-faceted strategy established here for hotspot evaluations and our insights into their mechanistic contributions are useful for modulating protein allostery in mechanistic and engineering studies.

Molecular and energetic basis for the broad allostery hotspots distribution in the tetracycline repressor

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