A-B-C is not as easy as 1-2-3 but assembles like clockwork
An experimental-computational approach develops a structurally stable model of the KaiABC complex and reveals a hitherto elusive mechanism in cyanobacterial circadian regulation
An international research team, including Kyoto University, ExCELLS, QST (Japan), the Institut Laue-Langevin, ILL (France) and others, has successfully established a novel integrated approach to solving the overall structure of the KaiABC complex in the cyanobacterial clock protein system.
The integrity of a biological system is maintained by its homeostatic activities involving dynamic assembly and disassembly of biomolecules, effectively its circadian rhythm. In cyanobacterial circadian regulation, three key proteins, KaiA, KaiB, and KaiC assemble to form the KaiABC complex. Cryogenic electron microscopy studies can identify most of the structure of this complex, but the KaiA protomers seem to be a bit too shy when researchers try to examine it microscopically.
Author Masaaki Sugiyama's team applied a combination of experimental techniques, including size-exclusion chromatography, small-angle X-ray scattering SEC-SAXS and inversed contrast matching small-angle neutron scattering -- SEC-iCM-SANS (Figure 1(a)). The aim was to obtain information on the location of KaiA protomers to feed into the analysis of the KaiABC complex's structure and dynamics.
The team computationally built 20 million structural models covering all possible KaiA conformations and then screened them out based on a set of experimental information. The surviving models were subjected to molecular dynamics simulations to examine their stability (Figure 1(b)). The final model obtained suggests that, despite large fluctuations in the KaiA N-terminal domains, their preferential positionings mask the hydrophobic surface of the KaiA C-terminal domain, hindering additional KaiA-KaiC interactions.
Many of the interesting proteins we would like to examine tend to form aggregates, preventing the extraction of molecular structure from the scattering curve. It is now possible to overcome this difficulty by using the Size-Exclusion Chromatography (SEC) set-up installed on the D22 small-angle diffractometer, to expose only the perfectly monodisperse samples exiting the column to the neutron beam, and record the resultant small-angle scattering curve.
This set-up is unique in the world, and it is thanks to the ILL's high-flux of neutrons that the data can be recorded before the molecules start to aggregate again. There are also other popular applications for the set-up, such as for buffer or detergent exchange.
The team's integrated approach provides a powerful and generally applicable tool for resolving masked structures of supramolecular complexes harbouring dynamically fluctuating domains or sub-units such as the KaiABC complex.
"SEC-SAXS and SEC-iCM-SANS were surprisingly crucial for structural screening," says Sugiyama. Co-author Atsushi Matsumoto adds, " Ten years ago it was just not feasible to cover all the KaiA conformations possible. Today the use of these methods accompanied by molecular dynamics simulations has become quite reasonable, thanks to the improved computational power available."
The study emphasizes that an integrated approach with multi-faceted techniques is essential in revealing the mysteries of biological phenomena.
Re.: “Overall structure of fully assembled cyanobacterial KaiABC circadian clock complex by an integrated experimental-computational approach", by Yasuhiro Yunoki et al. Commun Biol (2022).
The article can be accessed at https://doi.org/10.1038/s42003-022-03143-z