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Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Published on: September 27, 2012
Structural and Biophysical Basis of Circadian Output Kinase SasA and Its Discrete Stoichiometric Assembly With the
Sota W Suzuki1, Yuki Hirato2, Takahiro Iida2
1Graduate School of Science, Toho University, Funabashi, Chiba, Japan.
Abstract:
The cyanobacterial circadian clock is driven by a core post-translational oscillator composed of KaiA, KaiB, and KaiC, with the histidine kinase (HK), SasA, acting as the primary transducer of timing signals for downstream gene expression. Despite its central role, the structural architecture, oligomeric assembly of full-length SasA, and its stoichiometric interactions with the KaiC hexamer remain unclear. Herein, we determined the crystal structure of the SasA C-terminal ATPase domain (SasAC) from Thermosynechococcus vestitus BP-1 at 2.31 Å resolution. SasAC adopts a canonical GHKL family fold but features a distinct G2-box (ATP lid) orientation that differs markedly from other prototypical HKs. Mass photometry (MP) analysis of wild-type SasA and a truncated sensory domain mutant revealed that SasA undergoes concentration-dependent, even-numbered oligomerization (monomer-dimer-tetramer), establishing that SasA is predominantly a dimeric HK rather than the previously proposed trimeric or hexameric forms. Interaction assays demonstrated that the KaiC hexamer formed discrete complexes containing two or four SasA molecules, corresponding to 2:6 and 4:6 (SasA:KaiC) stoichiometries. These results provide a structural and biophysical framework for understanding SasA assembly with KaiC in the cyanobacterial circadian output pathway.
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