Abstract
The molecular recognition of the RORγ nuclear hormone receptor (NHR) ligand-binding domain (LBD) has been extensively studied with numerous X-ray crystal structures. However, the picture afforded by these complexes is static and does not fully explain the functional behavior of the LBD. In particular, the apo structure of the LBD seems to be in a fully active state, with no obvious differences to the agonist-bound structure. Further, several atypical in vivo inverse agonists have surprisingly been found to co-crystallize with the LBD in agonist mode (with co-activator), leading to a disconnection between molecular recognition and functional activity. Moreover, the experimental structures give no clues on how RORγ LBD binders access the interior of the LBD. To address all these points, we probe here, with a variety of simulation techniques, the fine structural balance of the RORγ LBD in its apo vs. holo form, the differences in flexibility and stability of the LBD in complex with agonists vs. inverse agonists and how binders diffuse in and out of the LBD in unbiased simulations. Our data conclusively point to the stability afforded by the so-called “agonist lock” between H479 and Y502 and the precise location of Helix 12 (H12) for the competence of the LBD to bind co-activator proteins. We observe the “water trapping” mechanism suggested previously for the atypical inverse agonists and discover a different behavior for the latter when co-activator is present or absent, which might help explain their conflicting data. Additionally, we unveil the same entry/exit path for agonists and inverse agonist into and out of the LBD for RORγ, suggesting it belongs to the type III NHR sub-family.
Highlights
On the other hand, its disruption leads to displacement or unfolding of Helix 12 (H12) and either co-repressor binding or no protein binding
A second matched molecular pairs (MMP) published by Biogen[6], with agonist compound 4 and inverse agonist compound 9 were included in the study to compare the effects agonist/inverse agonist have on the conformational rearrangements influencing the co-activator docking site
We reveal the RORγ apo structure, seemingly in the fully active state, features a H11/H11’/H12/co-activator area that fluctuates much more than an agonist-bound counterpart, leading to intermittent agonist lock breakage in solution
Summary
Its disruption leads to displacement or unfolding of H12 and either co-repressor binding (inverse agonists) or no protein binding (antagonists). It is accepted that ligands behaving as agonists must somehow stabilize the agonist lock and H12, whereas ligands behaving as antagonists and inverse agonists disrupt this interaction by directly H-bonding to H479, pushing H11 or disrupting the contact interface between helices H11, H11’ and H12 In spite of this simple theoretical description, the actual recognition landscape of the RORγ-LBD seems to be more complex than initially anticipated, probably involving dynamic aspects associated to the conformational transitions and the role of solvent that are not completely captured by experimental techniques. A common theme for this type of puzzling inverse agonists is the presence of water molecules inside the LBD in the vicinity of the H11-H12 interface, together with the bound ligand This has prompted to propose a “water trapping” mechanism[5], whereby these atypical inverse agonists behave as agonists in the crystal structures but might be unstable in vivo due to the destabilizing effect of trapped solvent molecules. A complete study of both agonists and inverse agonists accessing and exiting the same NHR is lacking
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