Technical Paper


Navigation Bar



Technical Paper

Tying it all together

Fun Facts


References and Additional Links

An analysis of:

Structure of the C-terminal domain of the clock protein KaiA in complex with a KaiC-derived peptide: Implications for KaiC regulation

Ionannis Vakonakis and Andy C. LiWang

With advising help from:

Dr. Megan McEvoy
Department of Biochemistry and Molecular Biophysics

It’s as easy as Kai A, B, C!

General Info about Cyanobacteria: (the organisms studied in this experiment) (definition)

  • Cyanobacteria are among the oldest organisms on Earth
  • They are very diverse: different species of cyanobacteria can survive in a broad range of environmental conditions
  • Even though cyanobacteria are unicellular, they perform many metabolic activities: photosynthesis, carbon fixation, and nitrogen fixation
  • Their circadian rhythms (which were also studied in this experiment) are among the oldest and simplest on Earth
  • Because they are easily amenable to genetic studies, they are ideal organisms to study


There are three genes that are responsible for the circadian rhythms in cyanobacteria: kaiA, kaiB, and kaiC, which code for the three proteins KaiA, KaiB, and KaiC, respectively

About the proteins:

  • KaiA has two subunits separated by a groove, and is a complex structure
  • KaiB is still being studied, especially the exact role it plays in circadian rhythm expression
  • KaiC is an autokinase (definition). Its phosphorylated and unphosphorylated forms correspond to the clock; however, this only occurs in equilibrium

It is believed that the interaction between KaiA and KaiC is the key to understanding how circadian rhythms work

LiWang and colleagues (authors of the report) looked at the structures of KaiA-KaiC complexes in vivo (definition) and in vitro (definition) to study the regions at which they interact.

Materials and Methods

The protein samples were prepared, and then their structures were analyzed by NMR (definition) (NMR website)


KaiC was expected to be composed of Rec-A like domains. RecA is a type of recombination protein that modifies DNA structure and function

KaiC peptides were also expected to pass through a groove in KaiA

The structure of KaiA: it is a 4 helix bundle with 2 subunits, which makes it a dimer (definition). It has a groove in the middle that is stabilized by electrostatic and hydrophobic hydrogen bonds (definitions).

KaiC peptides were found in the experiment to follow this middle groove (lie along it), which was different than the original hypothesis

KaiC attachment groove
Used with permission of Ionannis Vakonakis and Andy C. LiWang

It is necessary for KaiA to have a dimer structure because KaiC peptides interact with both of the KaiA subunits

The angle of dimerization (which in English means the angle formed between the two KaiA subunits) changes upon KaiC binding. Taken one step further, this means that KaiC changes the quarternary structure of the C-terminal of KaiA

Change in the angle of dimerization
Used with permission of Ionannis Vakonakis and Andy C. LiWang

So...what does all of this mean in terms of the circadian clock? Basically, the goal of this experiment was to study the interactions between KaiA and KaiC proteins and determine how these interactions play a role in the regulation of circadian clocks (if at all). After performing the experiment, the researchers discovered that the location of KaiA and KaiC interaction is important because it is the same location at which some substituted amino acids cause changes in the clock period. In plainer terms, the amino acid substitutions weaken the interactions between KaiA and KaiC, which lengthens the clock.


Protein interactions (possibly due to the CikA, which is a sensory protein that starts a protein cascade) result in changes in KaiA, which affect its affinity for KaiC

Signal Transduction pathway
Graphic Recreated from Susan S. Golden 2003.

Since KaiA enhances the ability of KaiC to phosphorylate itself (autokinase activity), this regulates the clock period

There is more KaiC in vivo than KaiA; therefore it is proposed that each KaiA binds to 2 KaiC molecules. This in turn may indicate that KaiA is a linker module that allows 2 KaiCs to transphosphorylate


University of Arizona
Biology 181H Group 03
Site Manager
Revised: 13 Nov 2004
Group 03 Home