Structural Superimposition

Abstact

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 (SasA(C)) from Thermosynechococcus vestitus BP-1 at 2.31 A resolution. SasA(C) 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.