A parametrized two-domain thermodynamic model explains diverse mutational effects on protein allostery

Reviewer #1 (Public Review):

Summary:

The authors' earlier deep mutational scanning work observed that allosteric mutations in TetR (the tetracycline repressor) and its homologous transcriptional factors are distributed across the structure instead of along the presumed allosteric pathways as commonly expected. Especially, in addition, the loss of the allosteric communications promoted by those mutations, was rescued by additional distributed mutations. Now the authors develop a two-domain thermodynamic model for TetR that explains these compelling data. The model is consistent with the in vivo phenotypes of the mutants with changes in parameters, which permits quantification. Taken together their work connects intra- and inter-domain allosteric regulation that correlate with structural features. This leads the authors to suggest broader applicability to other multidomain allosteric proteins.

Here the authors follow their first innovative observations with a computational model that captures the structural behavior, aiming to make it broadly applicable to multidomain proteins. Altogether, an innovative and potentially useful contribution.

Weaknesses:

None that I see, except that I hope that in the future, if possible, the authors would follow with additional proteins to further substantiate the model and show its broad applicability. I realize however the extensive work that this would entail.

Reviewer #2 (Public Review):

Summary:

This combined experimental-theoretical paper introduces a novel two-domain statistical thermodynamic model (primarily Equation 1) to study allostery in generic systems but focusing here on the tetracycline repressor (TetR) family of transcription factors. This model, building on a function-centric approach, accurately captures induction data, maps mutants with precision, and reveals insights into epistasis between mutations.

Strengths:

The study contributes innovative modeling, successful data fitting, and valuable insights into the interconnectivity of allosteric networks, establishing a flexible and detailed framework for investigating TetR allostery. The manuscript is generally well-structured and communicates key findings effectively.

Weaknesses:

The only minor weakness I found was that I still don't have a better sense into (a) intuition and (b) mathematical derivation of Equation 1, which is so central to the work. I would recommend that the authors provide this early on in the main text.

Reviewer #3 (Public Review):

Summary:

Allosteric regulations are complicated in multi-domain proteins and many large-scale mutational data cannot be explained by current theoretical models,, especially for those that are neither in the functional/allosteric sites nor on the allosteric pathways. This work provides a statistical thermodynamic model for a two-domain protein, in which one domain contains an effector binding site and the other domain contains a functional site. The authors build the model to explain the mutational experimental data of TetR, a transcriptional repress protein that contains a ligand and a DNA-binding domain. They incorporate three basic parameters, the energy change of the ligand and DNA binding domains before and after binding, and the coupling between the two domains to explain the free energy landscape of TetR's conformational and binding states. They go further to quantitatively explain the in vivo expression level of the TetR-regulated gene by fitting into the induction curves of TetR mutants. The effects of most of the mutants studied could be well explained by the model. This approach can be extended to understand the allosteric regulation of other two-domain proteins, especially to explain the effects of widespread mutants not on the allosteric pathways.

Strengths:

The effects of mutations that are neither in the functional or allosteric sites nor in the allosteric pathways are difficult to explain and quantify. This work develops a statistical thermodynamic model to explain these complicated effects. For simple two-domain proteins, the model is quite clean and theoretically solid. For the real TetR protein that forms a dimeric structure containing two chains with each of them composed of two domains, the model can explain many of the experimental observations. The model separates intra and inter-domain influences that provide a novel angle to analyse allosteric effects in multi-domain proteins.

Weaknesses:

As mentioned above, the TetR protein is not a simple two-main protein, but forms a dimeric structure in which the DNA binding domain in each chain forms contacts with the ligand-binding domain in the other chain. In addition, the two ligand-binding domains have strong interactions. Without considering these interactions, especially those mutants that are on these interfaces, the model may be oversimplified for TetR.