Atomistic simulations shed new light on the activation mechanisms of RORγ and classify it as Type III nuclear hormone receptor regarding ligand-binding paths

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.

RORγ Structural Plasticity and Druggability

“…It is now commonly accepted that RORγ orthosteric binders mainly regulate the transcriptional function by manipulating the stability of the lock. The results of molecular dynamics (MD) simulations further confirm it by showing lower fluctuations in interaction energy for the lock in the agonist-bound LBD and higher fluctuations for the lock in the unbound or inverse agonist-bound LBD [29]. Furthermore, they provide evidence for the existence of the lock in Figure 1.…”

“…Another in silico method, MD, has aided the study of the H11, H11,' H12 flexibility affected by different types of ligands and the atomic mechanisms on activating or inactivating RORγ and other NRs [29,53,54]. It is also an alternative method to study the SAR for a protein-ligand complex that lacks a co-crystal structure.…”

RORγ Structural Plasticity and Druggability

“…It is now commonly accepted that RORγ orthosteric binders mainly regulate the transcriptional function by manipulating the stability of the lock. The results of molecular dynamics (MD) simulations further confirm it by showing lower fluctuations in interaction energy for the lock in the agonist-bound LBD and higher fluctuations for the lock in the unbound or inverse agonistbound LBD [25]. Furthermore, they provide evidence for the existence of the lock in the unbound state, even if energetically unfavorable, implying that the H12 conformation in the unbound LBD crystal could be one of many conformations switching in solution.…”

Monte Carlo simulations using PELE to identify a protein–protein inhibitor binding site and pose

“…Early reports involving the study of cytochrome P450s, myoglobin or fatty acid binding protein 19,40 have now been complemented with multiple recent studies involving kinases, GPCRs, HIV-1 protease, epoxide hydrolase and various nuclear hormone receptors. 31,32,36,[41][42][43][44][45][46] Thus the application of PELE as a reliable induced-t binding tool has been amply proven. We show here for the rst time, the application of PELE in the context of protein-protein disruptor design, where many in silico methodologies struggle.…”