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Journal of Bacteriology, August 1999, p. 4628-4638, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

MgATP Binding and Hydrolysis Determinants of NtrC, a Bacterial Enhancer-Binding Protein

Irene Rombel,1,dagger Petra Peters-Wendisch,1 Andrew Mesecar,2,Dagger Thorgeir Thorgeirsson,3,§ Yeon-Kyun Shin,3 and Sydney Kustu1,*

Department of Plant and Microbial Biology,1 Department of Molecular and Cell Biology,2 and Department of Chemistry,3 University of California, Berkeley, California 94720

Received 9 April 1999/Accepted 24 May 1999

When phosphorylated, the dimeric form of nitrogen regulatory protein C (NtrC) of Salmonella typhimurium forms a larger oligomer(s) that can hydrolyze ATP and hence activate transcription by the sigma 54-holoenzyme form of RNA polymerase. Studies of Mg-nucleoside triphosphate binding using a filter-binding assay indicated that phosphorylation is not required for nucleotide binding but probably controls nucleotide hydrolysis per se. Studies of binding by isothermal titration calorimetry indicated that the apparent Kd of unphosphorylated NtrC for MgATPgamma S is 100 µM at 25°C, and studies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 µM at 37°C. Filter-binding studies with mutant forms of NtrC defective in ATP hydrolysis implicated two regions of its central domain directly in nucleotide binding and three additional regions in hydrolysis. All five are highly conserved among activators of sigma 54-holoenzyme. Regions implicated in binding are the Walker A motif and the region around residues G355 to R358, which may interact with the nucleotide base. Regions implicated in nucleotide hydrolysis are residues S207 and E208, which have been proposed to lie in a region analogous to the switch I effector region of p21ras and other purine nucleotide-binding proteins; residue R294, which may be a catalytic residue; and residue D239, which is the conserved aspartate in the putative Walker B motif. D239 appears to play a role in binding the divalent cation essential for nucleotide hydrolysis. Electron paramagnetic resonance analysis of Mn2+ binding indicated that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.

* Corresponding author. Mailing address: 111 Koshland Hall, U.C. Berkeley, Berkeley, CA 94720-3102. Phone: (510) 643-9308. Fax: (510) 642-4995. E-mail: kustu{at}nature.berkeley.edu.

dagger Present address: U.T. Southwestern Medical Center, Department of Internal Medicine and Cardiology, Dallas, TX 75235-8573.

Dagger Present address: Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607.

§ Present address: deCODE Genetics, Inc., 110 Reykjavik, Iceland.

Journal of Bacteriology, August 1999, p. 4628-4638, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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