Insight into cyanobacterial circadian timing from structural details of the KaiB–KaiC interaction

Significance The Kai system is a widely studied model in theoretical biology and systems biology. It is to date the only known circadian clock that can be reconstituted in vitro. Essential to the rhythmicity of the system is the formation of the KaiC–KaiB complex. Many aspects of this interaction, such as the mode of binding of KaiB, the stoichiometry of the interaction, and the exact binding interfaces have long remained ambiguous. We present a mass spectrometry-based structural model of the KaiC–KaiB interaction that answers many of these outstanding questions on the basis of direct experimental evidence. This structural model sheds light on the intricate workings of the in vitro oscillator. Circadian timing in cyanobacteria is determined by the Kai system consisting of KaiA, KaiB, and KaiC. Interactions between Kai proteins change the phosphorylation status of KaiC, defining the phase of circadian timing. The KaiC–KaiB interaction is crucial for the circadian rhythm to enter the dephosphorylation phase but it is not well understood. Using mass spectrometry to characterize Kai complexes, we found that KaiB forms monomers, dimers, and tetramers. The monomer is the unit that interacts with KaiC, with six KaiB monomers binding to one KaiC hexamer. Hydrogen–deuterium exchange MS reveals structural changes in KaiC upon binding of KaiB in both the CI and CII domains, showing allosteric coupling upon KaiB binding. Based on this information we propose a model of the KaiB–KaiC complex and hypothesize that the allosteric changes observed upon complex formation relate to coupling KaiC ATPase activity with KaiB binding and to sequestration of KaiA dimers into KaiCBA complexes.

[1]  Alexandre M J J Bonvin,et al.  HADDOCK versus HADDOCK: New features and performance of HADDOCK2.0 on the CAPRI targets , 2007, Proteins.

[2]  E. Pai,et al.  Anabaena circadian clock proteins KaiA and KaiB reveal a potential common binding site to their partner KaiC , 2004, The EMBO journal.

[3]  K. Namba,et al.  Functionally Important Substructures of Circadian Clock Protein KaiB in a Unique Tetramer Complex* , 2005, Journal of Biological Chemistry.

[4]  Martin Egli,et al.  Elucidating the Ticking of an In Vitro Circadian Clockwork , 2007, PLoS biology.

[5]  Tetsuya Mori,et al.  Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K. Namba,et al.  The Roles of the Dimeric and Tetrameric Structures of the Clock Protein KaiB in the Generation of Circadian Oscillations in Cyanobacteria* , 2012, The Journal of Biological Chemistry.

[7]  C. Johnson,et al.  Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria. , 1998, Science.

[8]  A. LiWang,et al.  Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria. , 2014, Journal of molecular biology.

[9]  Michael J Rust,et al.  References and Notes Supporting Online Material Materials and Methods Figs. S1 to S8 Tables S1 to S3 References Ordered Phosphorylation Governs Oscillation of a Three-protein Circadian Clock , 2022 .

[10]  Andy LiWang,et al.  Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria , 2011, Proceedings of the National Academy of Sciences.

[11]  A. Heck.  Native mass spectrometry: a bridge between interactomics and structural biology , 2008, Nature Methods.

[12]  Tetsuya Mori,et al.  Combined SAXS/EM Based Models of the S. elongatus Post-Translational Circadian Oscillator and its Interactions with the Output His-Kinase SasA , 2011, PloS one.

[13]  C. Dominguez,et al.  HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. , 2003, Journal of the American Chemical Society.

[14]  T. Kondo,et al.  Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in Vitro , 2005, Science.

[15]  R. Pattanayek,et al.  Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator , 2013, Cell cycle.

[16]  Takao Kondo,et al.  KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system , 2003, The EMBO journal.

[17]  Takao Kondo,et al.  In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB , 2010, FEBS letters.

[18]  Connie Phong,et al.  Robust and tunable circadian rhythms from differentially sensitive catalytic domains , 2012, Proceedings of the National Academy of Sciences.

[19]  Lars Konermann,et al.  Hydrogen exchange mass spectrometry for studying protein structure and dynamics. , 2011, Chemical Society reviews.

[20]  A. Bonvin,et al.  The HADDOCK web server for data-driven biomolecular docking , 2010, Nature Protocols.

[21]  Tetsuya Mori,et al.  Intramolecular Regulation of Phosphorylation Status of the Circadian Clock Protein KaiC , 2009, PloS one.

[22]  Albert J R Heck,et al.  Ion mobility mass spectrometry of proteins and protein assemblies. , 2010, Chemical Society reviews.

[23]  Martin Egli,et al.  Visualizing a circadian clock protein: crystal structure of KaiC and functional insights. , 2004, Molecular cell.

[24]  Martin Egli,et al.  Analysis of KaiA–KaiC protein interactions in the cyano‐bacterial circadian clock using hybrid structural methods , 2006, The EMBO journal.

[25]  Martin Egli,et al.  Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Andy LiWang,et al.  The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria , 2008, Proceedings of the National Academy of Sciences.

[27]  T. Kondo,et al.  Circadian Autodephosphorylation of Cyanobacterial Clock Protein KaiC Occurs via Formation of ATP as Intermediate* , 2012, The Journal of Biological Chemistry.

[28]  Tetsuya Mori,et al.  Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC , 2003, The EMBO journal.

[29]  Tetsuya Mori,et al.  Intermolecular associations determine the dynamics of the circadian KaiABC oscillator , 2010, Proceedings of the National Academy of Sciences.

[30]  T. Kondo,et al.  Three major output pathways from the KaiABC-based oscillator cooperate to generate robust circadian kaiBC expression in cyanobacteria , 2010, Proceedings of the National Academy of Sciences.

[31]  J. Benesch.  Collisional activation of protein complexes: Picking up the pieces , 2009, Journal of the American Society for Mass Spectrometry.

[32]  T. Kondo,et al.  Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro. , 2006, Molecular cell.

[33]  Toshifumi Takao,et al.  A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria , 2007, The EMBO journal.

[34]  Albert J R Heck,et al.  A sequestration feedback determines dynamics and temperature entrainment of the KaiABC circadian clock , 2010, Molecular systems biology.

[35]  Takao Kondo,et al.  No Transcription-Translation Feedback in Circadian Rhythm of KaiC Phosphorylation , 2005, Science.

[36]  S. Golden,et al.  Simplicity and complexity in the cyanobacterial circadian clock mechanism. , 2010, Current opinion in genetics & development.

[37]  T. Kondo,et al.  Cyanobacterial daily life with Kai-based circadian and diurnal genome-wide transcriptional control in Synechococcus elongatus , 2009, Proceedings of the National Academy of Sciences.

[38]  Takao Kondo,et al.  ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria , 2007, Proceedings of the National Academy of Sciences.

[39]  Katsumi Imada,et al.  ATP‐induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC , 2003, Genes to cells : devoted to molecular & cellular mechanisms.

[40]  Qiang Wang,et al.  Elevated ATPase Activity of KaiC Applies a Circadian Checkpoint on Cell Division in Synechococcus elongatus , 2010, Cell.

[41]  Martin Egli,et al.  CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates that KaiB monomers interact with KaiC and block ATP binding clefts. , 2013, Journal of molecular biology.

[42]  Fumio Hayashi,et al.  Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC. , 2006, Biochemical and biophysical research communications.

[43]  Michael J Rust,et al.  Light-Driven Changes in Energy Metabolism Directly Entrain the Cyanobacterial Circadian Oscillator , 2011, Science.

[44]  Yuichiro Maéda,et al.  Assembly and disassembly dynamics of the cyanobacterial periodosome. , 2008, Molecular cell.

[45]  A. Heck,et al.  The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument. , 2001, Rapid communications in mass spectrometry : RCM.

[46]  Brandon T Ruotolo,et al.  Ion mobility–mass spectrometry analysis of large protein complexes , 2008, Nature Protocols.

[47]  Martin Egli,et al.  Structural model of the circadian clock KaiB–KaiC complex and mechanism for modulation of KaiC phosphorylation , 2008, The EMBO journal.

[48]  Yuichiro Maéda,et al.  Tracking and visualizing the circadian ticking of the cyanobacterial clock protein KaiC in solution , 2011, The EMBO journal.

[49]  Martin Egli,et al.  Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism. , 2012, Biochemistry.

[50]  Kenichi Hitomi,et al.  Tetrameric Architecture of the Circadian Clock Protein KaiB , 2005, Journal of Biological Chemistry.

[51]  Stefanie Hertel,et al.  Revealing a Two-Loop Transcriptional Feedback Mechanism in the Cyanobacterial Circadian Clock , 2013, PLoS Comput. Biol..

[52]  A. LiWang,et al.  Rhythmic ring–ring stacking drives the circadian oscillator clockwise , 2012, Proceedings of the National Academy of Sciences.

[53]  C. Robinson,et al.  Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. , 2010, Analytical chemistry.

[54]  T. Kondo,et al.  Nonparametric entrainment of the in vitro circadian phosphorylation rhythm of cyanobacterial KaiC by temperature cycle , 2009, Proceedings of the National Academy of Sciences.

[55]  Hanspeter Herzel,et al.  Functioning and robustness of a bacterial circadian clock , 2007, Molecular systems biology.