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J. Biol. Chem., Vol. 282, Issue 19, 14018-14027, May 11, 2007

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ATP Binding to the KTN/RCK Subunit KtrA from the K⁺-uptake System KtrAB of Vibrio alginolyticus

ITS ROLE IN THE FORMATION OF THE KtrAB COMPLEX AND ITS REQUIREMENT IN VIVO^*

From the Department of Microbiology, University of Osnabrück, D-49076 Osnabrück, Germany

Subunit KtrA of the bacterial Na⁺-dependent K⁺-translocating KtrAB systems belongs to the KTN/RCK family of regulatory proteins and protein domains. They are located at the cytoplasmic side of the cell membrane. By binding ligands they regulate the activity of a number of K⁺ transporters and K⁺ channels. To investigate the function of KtrA from the bacterium Vibrio alginolyticus (VaKtrA), the protein was overproduced in His-tagged form (His¹⁰-VaKtrA) and isolated by affinity chromatography. VaKtrA contains a G-rich, ADP-moiety binding ---fold ("Rossman fold"). Photocross-linking and flow dialysis were used to determine the binding of [³²P]ATP and [³²P]NAD⁺ to His¹⁰-VaKtrA. Binding of other nucleotides was estimated from the competition by these compounds of the binding of the ³²P-labeled nucleotides to the protein. [-³²P]ATP bound with high affinity to His¹⁰-VaKtrA (K_D of 9 µM). All other nucleotides tested exhibited K_D (K_i) values of 30 µM or higher. Limited proteolysis with trypsin showed that ATP was the only nucleotide that changed the conformation of VaKtrA. ATP specifically promoted complex formation of VaKtrA with the His-tagged form of its K⁺-translocating partner, VaKtrB-His⁶, as detected both in an overlay experiment and in an experiment in which VaKtrA was added to VaKtrB-His⁶ bound to Ni²⁺-agarose. In intact cells of Escherichia coli both a high of membrane potential and a high cytoplasmic ATP concentration were required for VaKtrAB activity. C-terminal deletions in VaKtrA showed that for in vivo activity at least 169 N-terminal amino acid residues of its total of 220 are required and that its 40 C-terminal residues are dispensable.

Received for publication, September 25, 2006 , and in revised form, February 20, 2007.

^* This work was supported by the Deutsche Forschungsgemeinschaft SFB431, Teilprojekt P6. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material.

This work is dedicated to E. C. Slater on the occasion of his 90th birthday.

² Present address: Institute of Biochemistry, University of Kiel, D-24098 Kiel, Germany.

³ Present address: Klinik und Poliklinik für Unfall-, Hand und Wiederherstellungschirurgie, University Clinics, University of Münster, D-48149, Münster, Germany.

¹ To whom correspondence should be addressed: Dept. of Microbiology, University of Osnabrück, D-49076 Osnabrück, Germany. Tel.: 541-969-2274; Fax: 541-696-2870; E-mail: kroening{at}biologie.uni-osnabrueck.de.

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