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J. Biol. Chem., Vol. 282, Issue 36, 26401-26408, September 7, 2007

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Structural and Functional Studies of the HAMP Domain of EnvZ, an Osmosensing Transmembrane Histidine Kinase in Escherichia coli^*

Ryuta Kishii, Liliana Falzon, Takeshi Yoshida, Hiroshi Kobayashi, and Masayori Inouye¹

From the Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd., Shimotsuga, Tochigi 329-0114, Japan and Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

The HAMP domain plays an essential role in signal transduction not only in histidine kinase but also in a number of other signal-transducing receptor proteins. Here we expressed the EnvZ HAMP domain (Arg¹⁸⁰–Thr²³⁵) with the R218K mutation (termed L_RK) or with L_RK connected with domain A (Arg¹⁸⁰–Arg²⁸⁹) (termed LA_RK) of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli, by fusing it with protein S. The L_RK and LA_RK proteins were purified after removing protein S. The CD analysis of the isolated L protein revealed that it consists of a random structure or is unstructured. This suggests that the EnvZ HAMP domain by itself is unable to form a stable structure and that this structural fragility may be important for its role in signal transduction. Interestingly the substitution of Ala¹⁹³ in the EnvZ HAMP domain with valine or leucine in Tez1A1, a chimeric protein of Tar and EnvZ, caused a constitutive OmpC phenotype. The CD analysis of LA_RK(A193L) revealed that this mutated HAMP domain possesses considerable secondary structures and that the thermostability of this entire LA_RK(A193L) became substantially lower than that of LA_RK or just domain A, indicating that the structure of the HAMP domain with the A193L mutation affects the stability of downstream domain A. This results in cooperative thermodenaturation of domain A with the mutated HAMP domain. These results are discussed in light of the recently solved NMR structure of the HAMP domain from a thermophilic bacterium (Hulko, M., Berndt, F., Gruber, M., Linder, J. U., Truffault, V., Schultz, A., Martin, J., Schultz, J. E., Lupas, A. N., and Coles, M. (2006) Cell 126, 929–940).

Received for publication, February 15, 2007 , and in revised form, July 13, 2007.

^* This work was supported by National Institutes of Health Grant GM076587A. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-4115; Fax: 732-235-4559; E-mail: inouye{at}umdnj.edu.

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