While allostery has been a topic of intense interest for the past several decades, our understanding of the underlying mechanism at the molecular level continues to be challenged by new experimental observations. Specifically, a recent deep mutational scanning study of a bacterial transcription factor TetR found that allostery hotspot residues are broadly distributed over a major portion of the protein structure, rather than being clustered near the ligand-binding and DNA-binding domain interfaces as often discussed in structure-based studies. Similarly, loss of inducibility due to mutation of hotspots could be rescued by additional mutations that were also broadly distributed throughout the protein. These findings suggest that the contributions of hotspot residues are unlikely explained by a single mechanism, thus calling for different analysis strategies compared to previous computational studies. Motivated by these considerations, we have conducted extensive molecular dynamics simulations and free energy calculations for different ligation states of TetR. The computed free energy landscapes explicitly illustrate that allostery in TetR is well described by a conformational selection model, in which the apo state samples a broad set of conformations, and specific ones are selectively stabilized by either ligand or DNA binding. By examining a range of structural and dynamic properties of residues at both local and global scales, we find that various computational analyses capture different subsets of experimentally identified hotspots, supporting that these residues modulate allostery in distinct ways. These computational results motivate the development of an MWC-like thermodynamic model that qualitatively explains the broad distribution of hotspot residues and their distinct features in molecular dynamics simulations. The key realization is that allostery hotspots may contribute by either mediating inter-domain communications or intra-domain energetics. Thus our analysis highlights that the ``communication and ``shifting ensemble'' views of protein allostery are not in conflict, since both types of allostery hotspots are likely to contribute in a single system, which explains their broad distribution.
