Functionally Important Substructures of Circadian Clock Protein KaiB in a Unique Tetramer Complex

Katsumi Imada,Yukio Furukawa,Keiichi Namba,Fumio Hayashi,Masahiro Ishiura,Kiyoshi Onai,Tatsuya Uzumaki,Megumi Morishita,Ryo Iwase

doi:10.1074/jbc.m503360200

Abstract

KaiB is a component of the circadian clock molecular machinery in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. Here we report the x-ray crystal structure of KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The KaiB crystal diffracts at a resolution of 2.6 A and includes four subunits organized as a dimer of dimers, each composed of two non-equivalent subunits. The overall shape of the tetramer is an elongated hexagonal plate, with a single positively charged cleft flanked by two negatively charged ridges whose surfaces includes several terminal chains. Site-directed mutagenesis of Synechococcus KaiB confirmed that alanine substitution of residues Lys-11 or Lys-43 in the cleft, or deletion of C-terminal residues 95-108, which forms part of the ridges, strongly weakens in vivo circadian rhythms. Characteristics of KaiB deduced from the x-ray crystal structure were also confirmed by physicochemical measurements of KaiB in solution. These data suggest that the positively charged cleft and flanking negatively charged ridges in KaiB are essential for the biological function of KaiB in the circadian molecular machinery in cyanobacteria.

Highlights

Proteins have been demonstrated in vivo and in the yeast two-hybrid system [3]
The structure of the N-terminal amplitude-amplifier domain of the KaiA dimer has been determined by NMR [10], and the structure of the C-terminal clock-oscillator domain has been determined by NMR [11] and x-ray crystallography [5, 12]
We report here the 2.6 Å x-ray crystal structure of T. elongatus KaiB, which reveals that KaiB is an unusual tetramer, composed of two asymmetrical dimers, that assumes the shape of an elongated hexagonal plate

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—T. elongatus KaiB and KaiB T64C were expressed and purified as described previously [19]. A total of 6 ␮g of KaiB was incubated at 25 °C with 20 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Pierce) and 20 mM N-hydroxysulfosuccinimide (Pierce) in 20 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl. After 0, 15, 60, or 120 min, the reaction was terminated by the addition of 1 M Tris-HCl (pH 8.0) and 20 mM ␤-mercaptoethanol at final concentrations of 333 mM and 6.7 mM, respectively. Analytical Ultracentrifugation—The molecular weight of KaiB was determined by sedimentation equilibrium analysis at 20 °C on an Optima XL-A analytical ultracentrifuge (Beckman) with various rotation speeds, using 0.49 or 0.73 mg/ml KaiB in 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The molecular mass was analyzed using the program Optima TM XL-A/XLI version 4.0 (Beckman). In Vitro Mutagenesis, Gene Transfer, and in Vivo Rhythm Assay—We mutated kaiB genes by PCR-mediated site-directed in vitro mutagenesis

Summary of refinement statistics

RESULTS

DISCUSSION