Signaling Pathway in Two-component Regulatory System
Revealed by Combination of X-ray Crystallography
and Solution Scattering Technique
A sensor histidine kinase (HK) and a cognate
response regulator (RR) are protein elements of a
two-component regulatory system, which areubiquitously present in prokaryotes, fungi and plants.
The system is involved in cellular signal transduction
for homeostasis in response to sudden changes in theenvironment. HK is a single peptide consisting of
three domains, i.e., a sensor, a dimerization/histidinecontaining phosphotransfer (DHp), and ATP-bindingcatalytic (CA) domains from N- to C-termini (Fig. 1).
HK functionally acts as a dimer through the interactionof the DHp domain between each subunit. The
sensor domain of HK senses an environmental
change as an external signal, and then theconformational change induced upon signal receipt
would be transmitted to either activate or inactivate
the CA domain. The activated CA domain catalyzes
the phosphorylation of the special His residue at theDHp domain using ATP. The phospho-group is thentransferred to the special Asp in RR, and the resultant
phospho-RR can act as a transcriptional factor for
promoting the expression of target proteins.
Depending on external signals such as metal ions,amino acids, nitrogen compounds, light, and osmoticpressure among others, the sensor domains display avariety of structures. For example, the oxygen sensor
FixL contains a heme iron as an O2 binding site in thesensor domain, while the ethylene sensor ETR1 has atransmembrane sensor domain containing Cu as anethylene binding site. On the other hand, thestructures of the DHp and CA domains in HK and ofRR are essentially the same among a variety of twocomponent systems. This fact implies that theinterdomain or intermolecular signal transduction canbe achieved using a similar mechanism. However,there has been no report on the structures of theHK/RR complex, although the structures of eachseparated domain have been reported for many HKs
and RRs in the two-component regulatory system.
The structure of the HK/RR complex of thethermophilic bacterium Thermotoga martina wasdetermined by the combination of X-ray
crystallography (BL44B2) and solution scattering(BL45XU) technique [1]. The solution scattering forHK was measured with and without the sensor
domain, and in the presence and absence of RR(titration experiment). A comparison of the SAXS
parameters thus obtained (Table 1) showed that thesensor domain of HK is located on the molecular edgeof the HK dimer giving the longest dimension, and that
two molecules of RR can associate with the central
portion of the HK dimer with a dissociation constant(Kd) of 8 × 10-11 M-2. On the other hand, an electron
density map of the HK/RR complex was obtained at
4.2 Å resolution. On the basis of structural information
obtained from the SAXS analysis, the domains ofeach subunit of HK and RR could be unambiguously
assigned, as shown in Fig. 2. To our knowledge, thisis the first report on spacial arrangement in the twocomponent regulatory system.

Fig. 1. Schematic drawing of two-component regulatory system.

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Table 1. Structural parameters obtained from solution scattering experiments

The structure of the HK/RR complex, even at a lowresolution, can provide us clues to unveilinginterdomain and intermolecular signal transductions in
the two-component regulatory system. (i) The sensor
domain of one subunit comes in contact with the CA
domain of the same subunit, suggesting that thesignal could directly transfer from the sensor to the CA
domains in the cis manner. (ii) In the complex
structure, the active site of the CA domain of one
subunit is located about 25 Å away from thephosphorylation target His in the DHp domain of

another subunit, because His imidazole can bephosphorylated in the trans manner. Therefore, in thecourse of the catalytic reaction, the CA domain would
move by 25 Å to directly comes in contact with theDHp domain in the absence of RR [2]. (iii) RR
interacts with the HK sensor domain as well as its
DHp domain, implying that RR might affect thesensing capability of the sensor domain. Thesefindings significantly support previous experimental
results for the oxygen sensor system [3].

Fig. 2. Electron density map of HK/RR complex and assignment of domains. (a) side view and (b) top
view along dimerization axis. The arrows indicate the pathway of the signal transduction we proposed.

References

Seiji Yamada, Shuji Akiyama and Yoshitsugu Shiro*[1] S. Yamada, S. Akiyama, H. Sugimoto, H. Kumita,K. Ito, T. Fujisawa, H. Nakamura, Y. Shiro: J. Mol. Biol.
362 (2006) 123.
[2] A. Marina et al.: EMBO J. 24 (2005) 4247.
[3] H. Nakamura et al.: Proc. Natl. Acad. Sci. USA 101(2004) 2742.

SPring-8 / RIKEN*E-mail: yshiro@riken.jp33