SUN-154 - Dynamics of activation microswitches in follicle-stimulating hormone (FSHR) variants leading to premature ovarian insufficiency (POI), and conformational changes following interaction with the allosteric agonist CAN1405.

Receptor proteins often exist in multiple conformational states, influenced by the agonist. We have recently shown that plasma membrane expression of certain misfolded FSHR variants that lead to POI may be rescued by the small-molecule CAN1405 (Endocrinology, doi: 10.1210/endocr/bqag045). To extend our previous observations on the conformational changes provoked by CAN1405 on the wild-type (WT) FSHR and 2 pathogenic mutants (D408Y and I423T), we devised and refined a new in silico analysis by combining the transmembrane (TM)-TM helix distances and the active, partially active, and inactive conformations of the FSHR according to the A100 index, and analyzed the effects of CAN1405 on this collective variable. Changes were correlated with studies in HEK293 cells expressing the FSHR variants.

Methods: The initial coordinates of the FSHR were taken from the cryo-EM267 structure PDB:8I2G. Molecular dynamics simulations were run at 300 K across 3 independent replicates (R1-R3, 400 ns/each). The A100 index was optimized for predicting active (A100>50) and inactive (A100 < 0) structures. The model was originally tested on 268 known GPCR structures, so it was possible to differentiate between active and inactive structures. HEK293 cells expressing the WT FSHR or rescued FSHR mutants were exposed to FSH or CAN1405; signaling was assessed employing a cAMP CRE-luciferase reporter system.

Results: In the absence of CAN1405, the WT FSHR populated active, partially active, and inactive conformations, showing large TM helices flexibility: In R1 and R3 runs, the flexibility of TM6 was evident in the contours of TM6-TM3, TM6-TM5, TM6-TM4, and TM3-TM6, without transitioning to the inactive state. However, in run R2, the receptor transitioned from the active to inactive state through reposition of TMs 6 and 7. By contrast, FSHR mutants showed restricted dynamics around the active or partially active states. Significant changes in the dynamics of microswitches were observed when CAN1405 was inserted in the interhelical binding pocket of all FSHR variants: In this location, both mutants enhanced their dynamics as much as necessary to overcome the energy barrier between the active and inactive states, thereby becoming more flexible. All FSH variants showed a significant FSH-stimulated increase in CRE-luciferase activity (from 1.41-2.72 fold-change) after rescue by CAN1405. Interestingly, the CRE-luciferase response to CAN1405 was not significantly different from that elicited by FSH.

Conclusions: Analysis of microswitches dynamics identified changes in the flexibility of the TM domains of FSHR mutants provoked by CAN1405, leading to conformational changes similar to those induced by FSH. Studying the dynamics of microswitches in misfolded FSHRs is an important tool to identify favorable conformational changes induced by allosteric agonists and for designing new molecules useful in the treatment of POI.

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