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

Abstract

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.

Intricate Protein-Protein Interactions in the Cyanobacterial Circadian Clock

“…The HDX data reveal protection in areas that include the subunit interface of one KaiC ring, and collisional cross-sections together with computational simulations favor the CII binding mode (Fig. 1E) (44). The cryo-EM results were also complemented by molecular dynamics simulations that indicated a clear advantage of the CII interface in terms of the buried surface when compared with CI (30).…”

“…For one, B-loops are available on the outer surface of the CI ring, and accessing them does not necessitate ATPase activity. Moreover, computational simulations of KaiC 6 B 6 complexes with KaiB monomers located on either the CI or the CII end demonstrated a significantly enhanced buried surface for the latter interaction (30,44) (Fig. 2E).…”

“…Both KaiB and KaiB* appear to favor the dimeric over the tetrameric state (7,42,60). As demonstrated by native MS, the quaternary structure adopted by KaiB, tetramer, dimer, or monomer, is influenced by both protein concentration and temperature, with lower concentrations and reduced temperatures favoring the monomer (44). From KaiB crystal structures (no NMR solution structure of KaiB has been reported to date), it is not obvious why removing the C-terminal tail should favor the dimer.…”

“…Cryo-EM and HDX/MS in conjunction with modeling both place KaiB monomers at CII subunit interfaces, thereby covering ATP binding clefts (Figs. 1G and 2A) (30,44). Because KaiBC association is closely linked to the phosphorylation of Ser-431 (13,14), i.e.…”

“…E, side view of three KaiC subunits rendered in surface mode with blue patches indicating regions between subunits that are protected from HDX (left panel). HADDOCK-based models of the KaiC 6 B 6 complex with KaiB monomers bound to either the CII (central panel) or the CI ring (right panel) (44). The former is favored based on MS-based ion mobility spectrometry-derived collisional cross-section data.…”