Molecular dynamics simulations of nucleotide release from the circadian clock protein KaiC reveal atomic-resolution functional insights

Abstract

The cyanobacterial clock proteins KaiA, KaiB, and KaiC form a powerful system to study the biophysical basis of circadian rhythms, because an in vitro mixture of the three proteins is sufficient to generate a robust ∼24-h rhythm in the phosphorylation of KaiC. The nucleotide-bound states of KaiC critically affect both KaiB binding to the N-terminal domain (CI) and the phosphotransfer reactions that (de)phosphorylate the KaiC C-terminal domain (CII). However, the nucleotide exchange pathways associated with transitions among these states are poorly understood. In this study, we integrate recent advances in molecular dynamics methods to elucidate the structure and energetics of the pathway for Mg·ADP release from the CII domain. We find that nucleotide release is coupled to large-scale conformational changes in the KaiC hexamer. Solvating the nucleotide requires widening the subunit interface leading to the active site, which is linked to extension of the A-loop, a structure implicated in KaiA binding. These results provide a molecular hypothesis for how KaiA acts as a nucleotide exchange factor. In turn, structural parallels between the CI and CII domains suggest a mechanism for allosteric coupling between the domains. We relate our results to structures observed for other hexameric ATPases, which perform diverse functions.

ATP hydrolysis by KaiC promotes its KaiA binding in the cyanobacterial circadian clock system

“…Reciprocally, it is possible that KaiA pulls out the A-loop and, thereby, causes a microenvironmental rearrangement surrounding the nucleotide-binding site of the KaiC CII ring, facilitating ADP release from it. This is supported by a recently reported molecular dynamics simulation of the KaiC hexamer (Hong et al, 2018).…”

Myosin and Other Energy-Transducing ATPases: Structural Dynamics Studied by Electron Paramagnetic Resonance

“…(Alternatively, KaiA may pull out the tail directly.) This view supports an attractive possibility that the extension or leaping-out of the tail transmits into the inside cavity of the KaiC6mer through loop and reaches autophosphorylation and ATPase sites to promote autophosphorylation and ADP release [148,149] (see Figure 11A). (Ishii et al,unpublished [147]).…”

Bayesian modeling reveals metabolite‐dependent ultrasensitivity in the cyanobacterial circadian clock

“…KaiA binding to the CII domain (Kim et al, 2008;Pattanayek & Egli, 2015) stimulates KaiC autophosphorylation (Iwasaki et al, 2002;Williams et al, 2002;Kageyama et al, 2006). Recent work has shown that KaiA can bind to KaiC and act as a nucleotide exchange factor (Nishiwaki-Ohkawa et al, 2014) by facilitating conformational changes at the subunit interface that promote solvent exposure of the nucleotide-binding pocket (Hong et al, 2018). It is currently unclear whether this nucleotide exchange activity is responsible for all of KaiA's effect on KaiC or whether it alters the KaiC catalytic cycle in other ways (see Appendix for further analysis of this issue).…”