HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

Thank you for submitting your article "HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Philip Cole as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This interesting paper uses a combination of four complementary approaches to explore the activation mechanism of the nuclear receptor RORγ: segmental dynamics assessed by HDX; mutagenesis, comparison of various synthetic ligands, and crystal structures of three ligands in complex with the receptor.

Major strengths of the paper are the importance of the question been asked and the translatability of the findings to other nuclear receptors where differential responses to ligands are observed but for which mechanism is lacking. The data presented are generally rigorous and convincing. What is especially interesting is the correlation between the HDX data of different regions of the LBD.

A limitation of the study is that "activation" is defined in the context of a synthetic reporter system in transfected cells. There are no experiments to address the biological significance of the relationship between structure and activity reported. However, the reviewers and editor agree that the study sufficiently advances the field to justify publication in the absence of major additional work. Textual revisions are requested as outlined below.

Essential revisions:

1) One of the important conclusions that the authors reach is that the relatively high "basal" activity observed in vivo for RORγ is due to constitutive binding of an endogenous ligand which they term a silent agonist. This argument is based on a mutation that reduces the intrinsic basal activity of the receptor, makes the receptor refractory to activation by endogenous ligands, but still enables the receptor to respond to certain synthetic ligands. For me this is a plausible explanation, but not proof. The mutation could be responsible for two independent consequences, i.e. loss of intrinsic activity and loss of endogenous ligand binding. It is not proven that these are linked. This caveat should be acknowledged and discussed.

2) The authors present the two-step activation resulting from ligand binding as if they are the first to propose this. The concept that the ligand first stabilizes the global fold of the LBD and then repositions H12 has been understood in the field for many years and exemplified by human mutations that affect both processes. This should be acknowledged and discussed.