View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Texas A&M Repository NMR structure of the KaiC-interacting C-terminal domain of KaiA, a circadian clock protein: Implications for KaiA–KaiC interaction Ioannis Vakonakis*, Jingchuan Sun†‡, Tianfu Wu†, Andreas Holzenburg*†‡, Susan S. Golden†§, and Andy C. LiWang*¶ Departments of *Biochemistry and Biophysics and †Biology and ‡Microscopy and Imaging Center, Texas A&M University, College Station, TX 77843 Edited by Adriaan Bax, National Institutes of Health, Bethesda, MD, and approved December 5, 2003 (received for review August 27, 2003) KaiA is a two-domain circadian clock protein in cyanobacteria, necessary for phosphorylation. Similar to other pseudo-receiver acting as the positive element in a feedback loop that sustains the domains (16), KaiA activation most likely involves direct pro- oscillation. The structure of the N-terminal domain of KaiA is that tein–protein interactions of the N-terminal domain (with an of a pseudo-receiver, similar to those of bacterial response regu- as-yet-unidentified protein) that result in functional modulation lators, which likely interacts with components of the clock-reset- of the C-terminal effector domain. We postulated that KaiA is ting pathway. The C-terminal domain of KaiA is highly conserved a two-domain response regulator that acts as a timing input among cyanobacteria and enhances the autokinase activity of device to control the phase of the circadian period by modulating KaiC. Here we present the NMR structure of the C-terminal domain the KaiC phosphorylation state (9). of KaiA from the thermophilic cyanobacterium Thermosynecho- Here we report the solution structure of the Ϸ12-kDa KaiC- coccus elongatus BP-1. This domain adopts a novel all ␣-helical interacting C-terminal KaiA domain (residues 180–283, hence- homodimeric structure. Several mutations known to affect the forth named ThKaiA180C) from the thermophilic cyanobacte- period of the circadian oscillator are shown to be located at an rium Thermosynechococcus elongatus BP-1 (17), which grows exposed groove near the dimer interface. This NMR structure and Ͼ a 21-Å-resolution electron microscopy structure of the hexameric naturally at temperatures 50°C. The superior thermal stability KaiC particle allow us to postulate a mode of KaiA–KaiC interac- of this domain compared with the equivalent domain of KaiA tion, in which KaiA binds a linker region connecting two globular from S. elongatus (Fig. 5, which is published as supporting KaiC domains. information on the PNAS web site) enabled us to solve the structure at the physiological temperature of 50°C. We also determined the structure of S. elongatus KaiC by electron ircadian timekeeping oscillators and their accompanying Cmachinery comprise widespread regulatory mechanisms microscopy (EM) and single-particle reconstruction to a reso- that control the Ϸ24-h temporal pattern of diverse physiological lution of 21 Å. The structures raise interesting proposals for the processes, ranging from leaf movement to transcription regula- mode of KaiA–KaiC interaction. tion (1, 2). Circadian oscillators have been identified in virtually Materials and Methods all eucaryotic groups, including insects, fungi, plants, and mam- mals (3), and in cyanobacteria (4) and share mechanistic simi- Protein Preparation. Samples of S. elongatus KaiC and full-length larities, such as positive and negative transcription–translation KaiA were prepared as described (9). Expression and purifica- feedback loops (3). The cyanobacterial clock is the simplest and tion of ThKaiA180C are described elsewhere (18). NMR sam- potentially oldest (5) identified thus far, composed of three ples consisted of Ϸ1.3 mM protein in a buffer containing 20 mM interacting clock proteins, KaiA, KaiB, and KaiC, (4, 6) that form NaCl and 20 mM Na2HPO4, pH 7.07, at 23°C. Heterodimeric heteromultimeric complexes in vivo of sizes that oscillate in a (13C͞12C) samples of ThKaiA180C were prepared by mixing circadian manner (7). Transcription from practically all pro- equimolar amounts of oxidized 13C͞15N-enriched and -unen- moter regions in Synechococcus elongatus PCC 7942 is under riched protein in 6 M GdHCl͞50 mM NaCl͞20 mM Tris⅐HCl, pH circadian control, and transcription levels oscillate in phase or in 7.0, with the addition of 100 mM ␤-mercaptoethanol. Complete antiphase with transcription from the kai locus (4, 8). Indeed, reduction of the intermolecular disulfide bond was monitored by even promoters from other organisms, such as Escherichia coli, SDS͞PAGE. The protein was reconstituted by slow dilution with become rhythmically expressed in S. elongatus (8). a 20-fold excess volume of 50 mM NaCl͞20 mM Tris⅐HCl, pH 7.0, We previously reported that KaiA enhances the autokinase buffer and exchanged to the final NMR buffer and reoxidized. activity of KaiC in vitro (9). The KaiC phosphorylation state The final protein concentration of the heterodimeric samples BIOCHEMISTRY changes in vivo with a circadian pattern (10, 11), and ATP- was a 1.0 mM ThKaiA180C dimer (0.5 mM heterodimer plus binding by KaiC promotes formation of a hexameric KaiC 0.25 mM fully enriched homodimer plus 0.25 mM unenriched particle (12, 13), which is presumed to be the functional form of homodimer). Protein concentration was determined by absor- the protein. Evidence suggests that KaiC binds forked DNA bance at 280 nm by using a corrected extinction coefficient as when in the hexameric form (13). KaiC preparations have been suggested by Pace et al. (19). subjected to electron microscopic studies by two independent groups resulting in two structures (12, 13). KaiB is known to attenuate the KaiA-enhanced KaiC autokinase activity (9, 10, This paper was submitted directly (Track II) to the PNAS office. 14); however, no other information regarding its biochemical Abbreviations: NOE, nuclear Overhauser effect; EM, electron microscopy. function is presently available. Data Deposition: The solution structure has been deposited in the Protein Data Bank, KaiA is a two-domain protein. We previously showed that the www.rcsb.org (PBD ID codes 1Q6A and 1Q6B for the average minimized structure and the C-terminal KaiA domain alone is sufficient in vitro to enhance 25-structure ensemble, respectively). the autokinase activity of KaiC (9), whereas the N-terminal §To whom correspondence regarding circadian aspects should be addressed. E-mail: domain has no effect in that assay. The N-terminal domain of [email protected]. KaiA adopts the fold of a canonical response regulator receiver ¶To whom correspondence regarding structural aspects should be addressed. E-mail: domain (9, 15), although the primary sequence is dissimilar to [email protected]. that of receivers, and it lacks the conserved aspartyl residue © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0305516101 PNAS ͉ February 10, 2004 ͉ vol. 101 ͉ no. 6 ͉ 1479–1484 NMR Experiments. Sequential backbone and sidechain assign- primarily in the N-terminal pseudo-receiver domain; the C- ments of ThKaiA180C have been reported (18). All experiments terminal domain is highly conserved among all KaiA proteins were performed at 50°C with Varian Inova 600- and 500-MHz known to date (Fig. 7, which is published as supporting infor- spectrometers equipped with triple axis gradient probes. The mation on the PNAS web site). For instance, the S. elongatus and isomerization state of all proline residues was determined as T. elongatus N- and C-terminal domains share 30% and 62% trans (20). Stereospecific assignments of methylene and methyl identical residues, respectively. Furthermore, there are KaiA ␹ ␹ protons and 1 and leucine 2 angle determinations were pre- proteins that lack the N-terminal domain altogether (Anabaena formed as described (9). Interproton nuclear Overhauser effect sp. PCC 7120) or have only a truncated version (Nostoc punc- (NOE) distance restraints were obtained by 13C͞13C-edited 4D tiforme). The highly conserved C-terminal domain does not show NOESY (100-ms mixing time), 15N-edited 3D NOESY (100-ms significant sequence similarity to any other protein or derived mixing time), and 13C-edited͞12C-filtered 3D NOESY (21) domain from any organism studied. This simultaneous high (120-ms mixing time) spectra. Hydrogen bond restraints were degree of conservation in cyanobacteria and absence in other applied based on hydrogen exchange protection data collected organisms suggests that the C-terminal domain of KaiA serves by NMR and the existence of expected regular secondary a function specific to this system. structure NOEs (20). Backbone dynamics measurements (15N The C-terminal domain of the S. elongatus KaiA was unfa- 15 15 Ϫ1 T1, N T2, and N{ H} NOE) were performed and analyzed vorable for NMR structure determination because of limited as described (22). Spectrometer temperature was calibrated with aliphatic proton chemical shift dispersion and fast exchange of a methanol sample. amide protons. In addition, thermal denaturation experiments showed little cooperativity of unfolding (Fig. 5). In contrast, the NMR Structure Calculations. ␸ and ␺ dihedral angle values were C-terminal domain of KaiA from T. elongatus has greater derived from TALOS (23). Only residues for which all 10 predic- chemical shift dispersion and displays excellent cooperativity tions lie in the same region of the Ramachandran plot were used. (Fig. 5). Thus, we proceeded to solve the structure of the T. The XPLOR-NIH software package (24) was used for all stages of elongatus KaiA C-terminal domain, which by sequence identity NMR structure calculations. A distance-geometry simulating should be virtually equivalent to that of S. elongatus. annealing protocol in Cartesian space was used to generate an We previously showed that the N-terminal domain of S. initial set of structures from NOE and dihedral angle data. After elongatus KaiA is monomeric in solution even at high concen- inspection, the structures were refined by a simulated slow- trations (9). Full-length S. elongatus KaiA, however, elutes off a cooling process and a final minimization.