Phosphorylation-dependent Remodeling of the CLOCK/BMAL1/nucleosome Complex
- 1Bezmialem Vakif University
Abstract
Phosphorylation of the CLOCK/BMAL1 complex is a reversible post-translational modification that plays a central role in regulating circadian oscillations; however, its underlying mechanistic basis remains poorly understood. Although biochemical studies have shown that phosphorylation modulates CLOCK/BMAL1 binding to DNA, yet it remains unclear whether these effects are confined to local perturbation or also propagate through allosteric effects. Moreover, the influence of phosphorylation on histone dynamics and transcription factor-nucleosome interactions has not been systematically investigated. Here, we address these questions using atomistic trajectories obtained through backmapping of coarse-grained molecular dynamics simulations based on the recently resolved cryo-EM structure of the CLOCK/BMAL1 and nucleosome complex. We investigated three experimentally identified phosphorylation states: CLOCK bHLH S38/42 , BMAL1 bHLH S78 , and simultaneous phosphorylation of both proteins. Our results demonstrate that phosphorylation regulates CLOCK and BMAL1 asymmetrically. Whereas phosphorylation weakens the interaction of the modified bHLH domain with the E-box, BMAL1 phosphorylation simultaneously enhances DNA engagement by the CLOCK bHLH domain, an effect that persists in the doubly phosphorylated complex. Steered pulling simulations showed that phosphorylation of the CLOCK bHLH domain results in mechanical stability comparable to that of the unphosphorylated system. In contrast, BMAL1 phosphorylation appears to reduce the mechanical stability of the bHLH–DNA interaction. Beyond modulating DNA binding, phosphorylation remodels protein–histone interactions by altering contacts between the CLOCK PAS-B domain and histone H3 and between the BMAL1 PAS-A domain and the H2A/H2B acidic patch, while simultaneously rewiring residue-correlation and allosteric communication networks throughout the heterodimer. Importantly, phosphorylation increases the separation between the CLOCK HI loop and the H3 α1–L1 elbow, supporting a structural model in which phosphorylation provides a more permissive environment for CRY1 recruitment to the chromatin-bound CLOCK–BMAL1 complex, thereby facilitating displacement-type transcriptional repression. Collectively, our findings reveal that phosphorylation regulates the CLOCK–BMAL1 complex through coordinated remodeling of DNA binding, nucleosome interactions, and long-range allosteric communication, providing a mechanistic framework for circadian transcriptional repression and a foundation for the rational design of therapeutics targeting the molecular circadian clock.
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BibTeX
@misc{amairy2026phosphorylationdependent,
title = {{Phosphorylation-dependent Remodeling of the CLOCK/BMAL1/nucleosome Complex}},
author = {D. Amairy and H. Pekel and S. Gul and O. Sensoy},
howpublished = {bioRxiv},
year = {2026},
month = jun,
doi = {10.64898/2026.06.30.735537},
url = {https://www.biorxiv.org/content/10.64898/2026.06.30.735537v2},
note = {Public domain},
}
RIS
TY - UNPB
TI - Phosphorylation-dependent Remodeling of the CLOCK/BMAL1/nucleosome Complex
AU - D. Amairy
AU - H. Pekel
AU - S. Gul
AU - O. Sensoy
PY - 2026
DA - 2026/06/30
PB - bioRxiv
DO - 10.64898/2026.06.30.735537
UR - https://www.biorxiv.org/content/10.64898/2026.06.30.735537v2
AB - Phosphorylation of the CLOCK/BMAL1 complex is a reversible post-translational modification that plays a central role in regulating circadian oscillations; however, its underlying mechanistic basis remains poorly understood. Although biochemical studies have shown that phosphorylation modulates CLOCK/BMAL1 binding to DNA, yet it remains unclear whether these effects are confined to local perturbation or also propagate through allosteric effects. Moreover, the influence of phosphorylation on histone dynamics and transcription factor-nucleosome interactions has not been systematically investigated. Here, we address these questions using atomistic trajectories obtained through backmapping of coarse-grained molecular dynamics simulations based on the recently resolved cryo-EM structure of the CLOCK/BMAL1 and nucleosome complex. We investigated three experimentally identified phosphorylation states: CLOCK bHLH S38/42 , BMAL1 bHLH S78 , and simultaneous phosphorylation of both proteins. Our results demonstrate that phosphorylation regulates CLOCK and BMAL1 asymmetrically. Whereas phosphorylation weakens the interaction of the modified bHLH domain with the E-box, BMAL1 phosphorylation simultaneously enhances DNA engagement by the CLOCK bHLH domain, an effect that persists in the doubly phosphorylated complex. Steered pulling simulations showed that phosphorylation of the CLOCK bHLH domain results in mechanical stability comparable to that of the unphosphorylated system. In contrast, BMAL1 phosphorylation appears to reduce the mechanical stability of the bHLH–DNA interaction. Beyond modulating DNA binding, phosphorylation remodels protein–histone interactions by altering contacts between the CLOCK PAS-B domain and histone H3 and between the BMAL1 PAS-A domain and the H2A/H2B acidic patch, while simultaneously rewiring residue-correlation and allosteric communication networks throughout the heterodimer. Importantly, phosphorylation increases the separation between the CLOCK HI loop and the H3 α1–L1 elbow, supporting a structural model in which phosphorylation provides a more permissive environment for CRY1 recruitment to the chromatin-bound CLOCK–BMAL1 complex, thereby facilitating displacement-type transcriptional repression. Collectively, our findings reveal that phosphorylation regulates the CLOCK–BMAL1 complex through coordinated remodeling of DNA binding, nucleosome interactions, and long-range allosteric communication, providing a mechanistic framework for circadian transcriptional repression and a foundation for the rational design of therapeutics targeting the molecular circadian clock.
ER -