Abstract
CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) are both transcription factors of the circadian core loop in mammals. Recently published mouse CLOCK-BMAL1 bHLH (basic helix-loop-helix)-PAS (period-ARNT-single-minded) complex structure sheds light on the mechanism for heterodimer formation, but the structural details of the protein-DNA recognition mechanisms remain elusive. Here we have elucidated the crystal structure of human CLOCK-BMAL1 bHLH domains bound to a canonical E-box DNA. We demonstrate that CLOCK and BMAL1 bHLH domains can be mutually selected, and that hydrogen-bonding networks mediate their E-box recognition. We identified a hydrophobic contact between BMAL1 Ile80 and a flanking thymine nucleotide, suggesting that CLOCK-BMAL1 actually reads 7-bp DNA and not the previously believed 6-bp DNA. To find potential non-canonical E-boxes that could be recognized by CLOCK-BMAL1, we constructed systematic single-nucleotide mutations on the E-box and measured their relevant affinities. We defined two non-canonical E-box patterns with high affinities, AACGTGA and CATGTGA, in which the flanking A7-T7′ base pair is indispensable for recognition. These results will help us to identify functional CLOCK-BMAL1-binding sites in vivo and to search for clock-controlled genes. Furthermore, we assessed the inhibitory role of potential phosphorylation sites in bHLH regions. We found that the phospho-mimicking mutation on BMAL1 Ser78 could efficiently block DNA binding as well as abolish normal circadian oscillation in cells. We propose that BMAL1 Ser78 should be a key residue mediating input signal-regulated transcriptional inhibition for external cues to entrain the circadian clock by kinase cascade.
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Acknowledgements
We thank Dr Erquan Zhang (National Institute of Biological Sciences, Beijing, China) for critical reading of the manuscript and thoughtful discussions, Qin Cao for data collection, Pu Gao for the help of data processing, Xiaoxia Yu for the technical help of ultracentrifugation, Lorenzo Finci for language editing of the manuscript. This work was supported by the National Basic Research Program of China (973 program; 2011CB911103 to XDS), 985 and 211 grants from Peking University.
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Supplementary information, Figure S1
The 2Fo-Fc electron density map of DNA (contoured at 1.5 σ) calculated from the molecular replacement solution. (PDF 765 kb)
Supplementary information, Figure S2
(A) Gel filtration results showed that, individual BMAL1 bHLH appeared to be only homodimer whereas individual CLOCK bHLH existed as equilibrium between homodimer and homotetramer in solution. (PDF 258 kb)
Supplementary information, Figure S3
ITC measurements of DNA binding affinities in the presence of NAD cofactors. (PDF 254 kb)
Supplementary information, Figure S4
Titration of CLOCK-BMAL1 bHLH domains into tandem E-box DNA. (PDF 252 kb)
Supplementary information, Table S1
Summary of ITC data (PDF 242 kb)
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Wang, Z., Wu, Y., Li, L. et al. Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with E-box DNA. Cell Res 23, 213–224 (2013). https://doi.org/10.1038/cr.2012.170
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DOI: https://doi.org/10.1038/cr.2012.170
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