What Is the Mechanism of the Cyanobacterial Circadian Oscillator? Mike Rust O’Shea Group 10/10/2007 MCB 186 CIRCADIAN BIOLOGY

Acknowledgments Joe Markson Daniel Fisher Bill Lane, John Neveu, Bogden Budnik, Renee Robinson Erin O’Shea

No Rhythm of kaiA or kaiBC mRNA or Protein Levels in Constant Darkness Tomita et al. Science 307: (2005)

KaiC Phosphorylation Oscillates without Transcription/Translation Tomita et al. Science 307: (2005)

KaiA + KaiB + KaiC Oscillate in vitro KaiAKaiBKaiC ATP Nishiwaki et al. Science 308: (2005)

KaiA and KaiB modulate KaiC autophosphorylation ATP KaiB KaiA KaiC How does this balance give rise to stable oscillations rather than equilibrium?

Requirements for Stable Oscillation Time delay between phosphorylation and dephosphorylation Mechanism to synchronize KaiC molecules (nonlinearity) Each molecule may progress through a one-way cycle, but stochasticity will dephase them

How does the system store information about time of day? Total phosphorylation is insufficent because it traverses the same level twice in a cycle Some models have hypothesized long-lived conformational changes or kinetically trapped protein complexes

KaiC’s Two Phosphorylation Sites Are the two sites (S431/T432)really functionally equivalent? T S Hexameric ring, homology to RecA/DnaB family

Four Possible KaiC Phosphoforms T S U-KaiC T-KaiC S-KaiC ST-KaiC ST T S U SDS-PAGE bands identified by mass spectrometry

Want to Measure Phosphoform Distribution During Circadian Cycle Tecan pipetting robot (Bauer center) oil reaction SDS + EDTA

Cyclically Ordered Phosphorylation ST The phosphoform distribution provides a biochemical distinction between the rising and falling phases where total phosphorylation levels are identical.

Phosphoform Distribution Determines the Phase Can we construct a model in which all of these phase information is stored in the KaiC phosphorylation state? If so, the state should predict the initial phase of the clock rather than just reporting it.

Phosphoform Distribution Determines the Phase Can we construct a model in which all of these phase information is stored in the KaiC phosphorylation state? If so, the state should predict the initial phase of the clock rather than just reporting it. incubate with tagged KaiA to phosphorylate pulldown KaiA, let KaiC auto- dephosphorylat e IP

Phosphoform Distribution Determines the Phase Can we construct a model in which all of these phase information is stored in the KaiC phosphorylation state? If so, the state should predict the initial phase of the clock rather than just reporting it.

Partial Reactions U T ST S U T S KaiC alone KaiA + KaiC KaiA both promotes phosphorylation and inhibits dephosphorylation

Combining the patterns seen in partial reactions.... U T ST S KaiC alone U T ST S KaiA+KaiC yields a similar pattern to the full oscillating reaction! KaiA+KaiCKaiC only

Periodic Inactivation of KaiA? KaiA active KaiA inactive KaiC molecules could stay synchronized because they are all in equilibrium with the same pool of KaiA which is changing activity

Global KaiA Activity Varies During The Cycle Phosphorylation of test KaiC only occurs during rising phase of clock If KaiA is active, naive, unphosphorylated tagged KaiC should be phophorylated. Add 10% His-KaiC at various points in the cycle:

Global KaiA Activity Varies During The Cycle Falling phase is immediately reversed with excess KaiA Another possibility is that KaiC is committed to dephosphorylation in some KaiA-independent way. Spike in 5x KaiA during dephosphorylation phase

S-KaiC Abundance Correlates with KaiA Activity KaiA active KaiA inactive threshold?

Does a Specific KaiC Phosphoform Control Inhibition? KaiB preferentially interacts with S-KaiC IP KaiB during cycle

A Model for the Phosphorylation Cycle Can measure rate constants from partial reactions Phosphorylation rate depends hyperbolically on [KaiA]

S-KaiC Inhibition of KaiA D We assume that one S-KaiC monomer binds to KaiB and inactivates one dimer of KaiA. The rates are thus nonlinear functions of [S-KaiC] can measure from partial reactions

This Model Predicts Limit- Cycle Circadian Oscillation ST ~21 hour period

Q ualitative Picture of How Oscillations Occur

critical balance between S-KaiC and ST-KaiC

Role of Each Kai Protein in This Model KaiC is the central time-keeping element. Its two phosphorylation sites encode the information about circadian time. KaiA enhances KaiC phosphorylation and inhibits dephosphorylation. Global changes in KaiA activity synchronize the KaiC molecules. KaiB specifically senses the S431-P form of KaiC and inactivates KaiA, closing a feedback loop which allows KaiC to regulate its own phosphorylation.

Requirements Fulfilled in the Model Time delay due to fact that T432 is phosphorylated first, but inhibition does not occur until only S431 is phosphorylated. Synchronization arises because all KaiC molecules are in rapid equilibrium with the same pool of KaiA.

Unanswered Questions How do input pathways to the core oscillator affect resetting and entrainment? How does KaiC achieve kinetic preferences and slow enzymatic rates (~ 0.2 / hour)? What is the importance of the transcriptional control of kaiA and kaiBC? Could eukaryotic clocks use similar post-translational strategies?

Requirement for sharp change in rates as S-KaiC levels change Small changes in %S-KaiC can cause large changes in KaiA activity

Sequestration Achieves Switch-Like Behavior

Bistability in S-KaiC / ST- KaiC SST rising phase falling phase

Model