MgATP binding and hydrolysis determinants of NtrC, a bacterial enhancer-binding protein.
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 K(d) of unphosphorylated NtrC for MgATPgammaS is 100 microM at 25 degrees C, and studies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 microM at 37 degrees 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 p21(ras) 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 Mn(2+) binding indicated that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.
Rombel
I
I
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA.
Peters-Wendisch
P
P
Mesecar
A
A
Thorgeirsson
T
T
Shin
Y K
YK
Kustu
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001426:Bacterial Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051922:PII Nitrogen Regulatory Proteins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D051922:PII Nitrogen Regulatory Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D001224:Aspartic Acid; D000255:Adenosine Triphosphate