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.

Keywords: allosteric regulation; conformational asymmetry; enhanced sampling methods; free energy methods; homohexameric ATPases.

Fig. 1.

The CII nucleotide release pathway involves both local and global conformational changes. (A) KaiC is a homohexamer with active sites located at the subunit interfaces. The hexamer forms a double-ring architecture; the N-terminal ring is termed the CI domain, and the C-terminal ring is termed the CII domain. The secondary structures of subunits D and E are shown as yellow and green ribbon diagrams, respectively, while the other four subunits are represented by a white van der Waals surface. The CI–CII linker (residues 248–261) and the A-loop (residues 486–498) of subunits D and E are colored blue. (B) A β-hairpin (β_nt; residues 468–483) and an α-loop-α motif (αlα; residues 321–342) show the biggest displacements in the locally enhanced sampling simulations. The color represents the per-residue rmsd between initial and final protein backbone conformations, mapped onto the initial structure. (C) The string calculation reveals concerted motion of the six CII intersubunit angles during ADP dissociation at the subunit D–E active site. (Inset) A schematic of the CII domain, illustrating the intersubunit angle variables and the ADP–subunit distance variables employed in the simulation. (D) The nucleotide moves radially outward in the string pathway; the arrows indicate the movement of N6 and Pβ atoms in ADP. In A, B, and D, the Mg·ADP atoms are colored by element (C, cyan; N, blue; O, red; P, gold; H, white; Mg, pink).

Fig. 2.

Electrostatic interactions dominate the energetics of nucleotide release. (A) The free energy profile of the release process, calculated using umbrella sampling. B and C show the interaction between Mg∙ADP and key active-site residues. The solid lines show the coordination of the Mg²⁺ ion, and the dotted lines represent hydrogen-bond interactions. Colors are the same as in Fig. 1D. (B) A representative nucleotide-bound structure, from (8.5 Å, 60.2°) in A. (C) A representative structure from the region of rapid increase in free energy, specifically (10.4 Å, 64.0°) in A. (D) Alternative projection of the umbrella sampling data in A. The CVs are the subunit D–E angle and the electrostatic interaction energy between solvent and active-site residues K294, T295, E319, and D378.

Fig. 3.

PCA reveals large-scale conformational changes during nucleotide release. (A) PC1 represents the formation of a split washer structure in CII, and (B) PC2 represents a circular compression in both domains. In A and B, arrows show the Cα difference vectors between a pair of umbrella-sampling structures representing the extremes of motion along each PC, aligned to minimize the difference in the CII domain of subunit D. Structures in A are chosen to have similar PC2 values, and structures in B are chosen to have similar PC1 values, such that the differences shown approximate motions in only the PC of interest. (C) Conformations of the CII domain in the low PC1 (Left) and high PC1 (Right) structure, respectively. The side-by-side comparison indicates that the PC1 eigenvector points in the direction of increased helical pitch in the CII domain.

Fig. 4.

The A-loop (residues 486–498) is coupled to nucleotide release. (A) The A-loop (blue) of subunit D (yellow) samples an extended conformation. (B) The structure of the subunit D A-loop in buried and extended states can be quantified using the angle formed by the Cα atoms at residues 486, 490, and 496, which are represented as gray spheres. The values of this A-loop angle are 33.6° and 75.8° in the buried and extended states shown in B. The subunit D–E angle values in the buried and extended structures are 62.9° and 65.7°, respectively. (C) Projection of the umbrella sampling data onto the CV space consisting of the A-loop angle and the subunit D–E angle shows that a more extended subunit D A-loop conformation is correlated with a larger subunit D–E angle.

Fig. 5.

CI and CII share similar conformations. In A and B, Secondary structural elements identified by the simulations as important for nucleotide release from CII (A) and their homologs in CI (B) are colored in blue. In each case, the structure shown is the domain from the D subunit of the crystal structure (PDB ID code 3DVL).