Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Unexpected Integrator of the Cyanobacterial Circadian Clock


By: Carol A. Rouzer, VICB Communications
Published:  August 29, 2018



Variable affinity between S. elongatus KaiA and KaiC proteins stabilizes clock function as the relative protein concentrations vary.


Circadian clocks are endogenous biochemical pacemakers comprising a group of proteins that undergo a series of cyclic phosphorylations and dephosphorylations with a strikingly accurate and highly reproducible 24 h periodicity. They regulate a wide variety of processes from gene expression and metabolism to reproduction and behavior. Thus, their oscillations must remain stable under the full range of biochemical conditions that might exist in a cell throughout its lifetime. This has led to one of the great mysteries of circadian clock physiology - how is the rhythm maintained even when the relative quantities of the individual clock proteins change in response to varying conditions? Now, an answer to this question comes from the work of VICB member Carl Hirschie Johnson, his collaborators Takayuki Uchihashi (Nagoya University) and Toshio Ando (Kanazawa University), and their laboratories. They identify a key mechanism by which the Synechococcus elongatus circadian clock (Figure 1) maintains its rhythm in the face of wide variations in the quantities of its protein components [T. Mori, S. Sugiyama, et al., (2018) Nat. Commun., 9, 3245].



FIGURE 1. The KaiC protein (pink ovals) comprises a C1 and a C2 domain with C-terminal "tentacles".  It exists as a hexamer in solution. At the beginning of the cycling reaction, KaiA (blue octagon) repeatedly and rapidly interacts with KaiC's C-terminal tentacles, initiating and propagating the phosphorylation phase. Phosphorylation occurs first at threonine-432 (yellow stars) and then at serine-431 (orange stars). When KaiC becomes hyperphosphorylated, it binds KaiB (green hexagon). Then, the KaiB•KaiC complex binds KaiA, sequestering it from further interaction with KaiC's tentacles. At that point, KaiC initiates dephosphorylation, first at threonine-432, and then at serine-431, with resynthesis of ATP. When KaiC is hypophosphorylated, it releases KaiB and KaiA, thereby launching a new cycle.  Image adapted from C. H. Johnson, et al., (2011) Annu. Rev. Biophys., 40, 167.



The S. elongatus circadian clock is the most thoroughly studied and well understood of all circadian clocks. The clock comprises three proteins, KaiA, KaiB, and KaiC. KaiC, which possesses both autokinase and autophosphatase activities, undergoes a rhythmic cycle of phosphorylation followed by dephosphorylation, aided by interactions with KaiA and KaiB. In solution, KaiC exists as a hexamer that, when hypophosphorylated, interacts with KaiA through "tentacles" located at its C-terminus. This interaction stimulates KaiC's autokinase activity, leading to phosphorylation, first at threonine-432, then at serine-431. Gradually, KaiC becomes hyperphosphorylated and begins to interact, at its N-terminus, with KaiB. Bound KaiB then associates with KaiA and prevents it from stimulating the autokinase activity of KaiC. As a result, the autophosphatase activity predominates, and KaiC is dephosphorylated at threonine-432 followed by serine-431, with resynthesis of ATP. As KaiC becomes hypophosphorylated, it releases KaiA and KaiB, setting the stage for the beginning of another cycle (Figure 1). This robust series of oscillating reactions occurs in vitro with roughly 24 hour periodicity, requiring only the presence of the three proteins and ATP. In vivo, interaction of the oscillating complex with other proteins signals gene expression changes in the cell that result in the circadian regulation of nearly all of S. elongatus's biochemical functions.


Prior work had led to the hypothesis that the affinity of KaiA for the C-terminal tentacles of KaiC varies with KaiC's phosphorylation status. In particular, the hypothesis specified that the affinity of the KaiA-KaiC interaction is higher when KaiC is hypophosphorylated and decreases as the phosphorylation state increases, a phenomenon called phosphoform-dependent differential affinity (PDDA). If true, this mechanism could help explain how the circadian rhythm remains synchronized, by guaranteeing that unphosphorylated KaiC subunits would be the most likely to bind KaiA and be stimulated to undergo autophosphorylation. For their new studies, the researchers initiated a more detailed look at this hypothesis. They used computational methods to confirm that PDDA should help to stabilize clock oscillations, but the range of kinetic values (rate constants for KaiA's association with and dissociation from KaiC) in which this occurred was quite limited.


Up to this time, PDDA had only been studied from a theoretical perspective. The investigators realized that to fully test the hypothesis required experimental data. To obtain these data, they used high speed atomic force microscopy (HS-AFM), a technique that enabled them to image and quantify the dynamic interactions between KaiA and KaiC in real time. They found that if they attached KaiC to slides composed of unmodified mica, the protein bound to the slide via its C-terminal tentacles. This orientation prevented an interaction between the KaiC tentacles and KaiA. In contrast, when the researchers attached KaiC to slides comprising 3-aminopropyl-trietoxy silane-treated mica (AP-mica), they bound via their N-terminus, leaving the C-terminal tentacles free to interact with KaiA. In this orientation, the investigators could observe added KaiA molecules associating with and dissociating from the bound KaiC (Figure 2). Thus, the approach enabled direct measurements of the rates of these interactions.



FIGURE 2. (a) HS-AFM image of KaiC bound to bare mica. The C-terminal tentacles attach to the mica and are unavailable to bind to KaiA when it is added. Thus, the images in the absence (-KaiA) and presence (+KaiA) of added KaiA appear the same. (b) HS-AFM image of KaiC bound to AP-mica. The N-terminus of the molecule attaches to the AP-mica leaving the tentacles free to interact with KaiA, which is visible as bright yellow spheres in the +KaiA images. Image reproduced under the T. Mori, S. Sugiyama, et al., (2018) Nat. Commun., 9, 3245.


The researchers used site-directed mutagenesis to create KaiC proteins that mimicked its various phosphorylation states. An S431A/T432A double mutant, which could not be phosphorylated, mimicked hypophosphorylated KaiC. A protein containing S431A/T432E mutations served as a model of single phosphate addition during the phosphorylation cycle, whereas an S431D/T432E mutant mimicked hyperphosphorylation. Single phosphorylation during the dephosphorylation cycle was modeled by an S341D/T432A mutant. Using these proteins in their HS-AFM studies, the researchers were able to show that, indeed, the affinity of KaiA for the KaiC tentacles decreased with increasing phosphorylation. They went on to confirm this using wild-type KaiC and KaiA recovered from in vitro incubations in which all three clock proteins were undergoing rhythmic cycling.

With experimental measurements of phosphorylation-dependent changes in the on- and off-rates of the KaiA/KaiC interaction now available, the investigators used a Monte Carlo approach to develop a stochastic computational model of the circadian clock. The model, which they created in two versions – one that did and one that did not include PDDA – enabled them to directly explore the impact of PDDA on clock synchronization. The results confirmed that PDDA does, in fact, increase clock synchrony; however, when they incorporated their actual experimental KaiA/KaiC affinity values, the effect was so small as to be almost negligible. In contrast, they discovered a totally unexpected benefit of PDDA. Specifically, the model predicted that PDDA helps the clock to maintain its rhythm over a wide variation in the ratio of KaiA to KaiC concentration, and the researchers verified this prediction experimentally.

The investigators concluded that, as KaiA concentration increases relative to that of KaiC, PDDA helps to promote the formation of hyperphosphorylated KaiC. This is the form of the protein that sequesters KaiA, enabling it to compensate for excessive levels of that protein (Figure 3). Thus, PDDA helps to dampen the effects of fluctuating KaiA levels by modulating its rate and level of sequestration. Although these findings are specific to the S. elongatus clock, similarities in clock function across species suggests that the PDDA mechanism likely has a broad application. Further work will help to reveal the general importance of PDDA to circadian rhythms, including those in humans.




FIGURE 3. Mechanism by which PDDA helps to stabilize the circadian clock. Beginning with KaiC (green) in the hypophosphorylated state (left), affinity for interactions between KaiA (brown) and the C-terminal tentacles of KaiC is high. The KaiA/KaiC interaction promotes phosphorylation of KaiC, leading to a loss of affinity for KaiA. When KaiC is hyperphosphorylated, KaiB (lavender) binds, and the KaiA/KaiB complex then sequesters KaiA. As KaiA no longer interacts at the C-terminal tentacles of KaiC, dephosphorylation begins, ultimately returning KaiC to the hypophosphorylated stated. At this point, both KaiB and KaiA dissociate, and the cycle begins again. Image reproduced under the from T. Mori, S. Sugiyama, et al., (2018) Nat. Commun., 9, 3245.



View Nature Communications article: Revealing circadian mechanisms of integration and resilience by visualizing clock proteins working in real time







The Vanderbilt Institute of Chemical Biology, 896 Preston Building, Nashville, TN 37232-6304, phone 866.303 VICB (8422), fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2014 by Vanderbilt University Medical Center