Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

Abstract

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Fig 1.

Alignments of the sequences of KaiC, KaiC-P2, and KaiCI. Black backgrounds indicate identical residues. Shaded backgrounds indicate conserved substitutions (criteria defined by CLUSTAL X). His-6 tags for protein purification are underlined. Hyphens indicate gaps.

Fig 2.

KaiC and KaiC-P2 form homohexameric complexes. (A and B) Time-derivative sedimentation velocity analysis of KaiC and KaiC-P2. Plots of g(s*) vs. s*. The peak value of s* gives the sedimentation coefficient, and the standard deviation gives the diffusion coefficient, both corrected to water at 20°C. The combination of s and D gives an estimate of molecular weight. (A) Analytical ultracentrifugation of KaiC in the presence of 5 mM ATP, 40,000 rpm. s_20,w = 11.7S; D_20,w = 3.17F; R_s = 6.8 nm; M = 336 kg/mol. (B) Analytical ultracentrifugation of KaiC-P2 in the absence and presence of 5 mM ATP, 50,000 rpm. In the presence of ATP: s_20,w = 11.4S; D_20,w = 2.77F; R_s = 7.8 nm; M = 368 kg/mol, whereas in the absence of ATP: s_20,w = 3.61S; D_20,w = 5.57F; R_s = 3.8 nm; M = 58.0 kg/mol. (C) Gel filtration of KaiC (Upper) and KaiC-P2 (Lower) complexes on a Sephacryl S-300 HR column. Positions of standards are shown at the top of Upper (standards: BD = blue dextran, AF = apoferritin, BA = β-amylase, B = BSA, C = cytochrome c).

Fig 3.

Formation of hexameric rings by KaiC-P2 specifically depends on ATP. Purified KaiC-P2 was incubated with the indicated nucleotides in 25 mM Hepes⋅KOH, pH 7.5/50 mM KCl/1 mM DTT/0.5 mM EDTA/±5 mM MgCl₂ at 4°C for 2 h, and analyzed by nondenaturing PAGE (the gel did not contain nucleotides). Lane 1, no nucleotides (includes 5 mM MgCl₂); lane 2, 5 mM ATP excluding MgCl₂; lane 3, 5 mM ATP; lane 4, 5 mM GTP; lane 5, 5 mM dATP; lane 6, 5 mM ADP; lane 7, 0.1 mM ATP (lanes 3–7 include 5 mM MgCl₂). H, migration position of hexameric complexes; M, migration position of monomers.

Fig 4.

EM of ATP-dependent protein complexes formed by the KaiC protein. A and B show the KaiC protein from S. elongatus PCC 7942 in an ATP-containing solution. (A) Sample viewed with the negative staining procedure. Enlarged sections of this image are shown in Insets. (B) A more diluted sample viewed after preparation by positive staining. (C) Model for KaiC hexamers. Each monomer consists of an N-terminal (CI) and a C-terminal (CII) half. These monomers associate into a hexamer in which the monomers surround a central pore.

Fig 5.

EMSA of KaiC-P2 and KaiC binding to DNA. (A) Binding of KaiC-P2 to DNA. Lanes 1–8, radiolabeled forked DNA probe (≈5 fmol/20-μl reaction volume). Lanes 2–8 include purified KaiC-P2 (0.46 μg = 7.26 pmol)/20-μl reaction volume) and 1 mM AMP-PNP. Lanes 3–7 include unlabeled forked DNA probe at the following concentrations per 20-μl reaction volume: 5 fmol (lane 3), 10 fmol (lane 4), 25 fmol (lane 5), 250 fmol (lane 6), and 2.5 pmol (lane 7). Lane 8 includes 2.5 pmol unlabeled 60-mer poly dN random oligonucleotide (single-stranded DNA). Incubation at 53°C was for 30 min before loading on the gel. “P” marks the position of the unbound probe. (B) Binding of KaiC and KaiC-P2. Lanes 1–3, radiolabeled forked DNA probe (≈3 fmol/20-μl reaction volume) and 1 mM AMP-PNP. Lane 2 includes purified KaiC-P2 (0.46 μg = 7.3 pmol/20-μl reaction volume). Lane 3 includes purified KaiC (0.52 μg = 8.8 pmol/20-μl reaction volume). There was a 30-min incubation before loading on the gel at 53°C (lane 2) or at 30°C (lane 3). “P” marks the position of the unbound probe.