A-B-C is not as easy as 1-2-3 but assembles like clockwork

Kyoto University

An international research team, including Kyoto University, has successfully established a novel integrated approach to solve 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 into the KaiABC complex. Cryogenic electron microscopy studies can identify its structure for the most part, but the KaiA protomers in the complex appear a bit too shy to let researchers see through it in microscopic examination.

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. They aimedto get information about the locations of KaiA protomers that helped to analyze the KaiABC complex’s structure and dynamics.

The team computationally built 20 million structural models covering all possible KaiA conformation and then screened them out based on a set of experimental information. The survived models were subjected to molecular dynamics simulations to examine their stabilities.

The final model suggests that, despite large fluctuations of the KaiA N-terminal domains, their preferential positionings mask the hydrophobic surface of the KaiA C-terminal domain, hindering additional KaiA-KaiC interactions.

The team’s integrated approach provides a powerful and generally applicable tool for resolving masked structures of supramolecular complexes harboring dynamically fluctuating domains or subunits like the KaiABC complex.

“SEC-SAXS and SEC-iCM-SANS were surprisingly crucial for structural screening,” says Sugiyama.

Co-author Atsushi Matsumoto adds, “Currently, our methods work as a reasonable approach accompanied by molecular dynamics simulations due to improved computational power, compared to ten years ago when covering all possible KaiA conformations was not feasible.”

The study emphasizes that an integrated approach with multi-faceted techniques is essential in uncovering the mysteries of biological phenomena.

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