Molecular Dynamics Simulations of the Circadian Clock Protein Kaic Reveal Structural Insights into the Nucleotide Release and Circadian Timing Mechanisms

Abstract

The cyanobacterium S. elongatus keeps track of time partly through three soluble clock proteins KaiA, KaiB, and KaiC. These proteins 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-hour rhythm in the phosphorylation of KaiC. The nucleotide bound states of KaiC is important in controlling 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 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. In particular, releasing the nucleotide requires solvating the active site, which in turn requires widening the subunit interface leading to the active site. This conformational rearrangement is linked to the 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.