MgATP binding and hydrolysis determinants of NtrC, a bacterial enhancer-binding protein

Abstract

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.

FIG. 1

Domain structure of NtrC. NtrC is comprised of three functional domains: an amino-terminal regulatory domain (∼120 amino acids) that is phosphorylated at aspartate 54 (D54), a central catalytic domain (∼240 amino acids) that contains determinants for ATP binding and hydrolysis, as well as for oligomerization and transcriptional activation, and a carboxy-terminal domain (∼90 amino acids) that contains a helix-turn-helix DNA-binding motif and dimerization determinants. Residues within the central catalytic domain of NtrC that appear to be required for MgATP binding (this work) are indicated by b, whereas residues that appear to be required for ATP hydrolysis but not binding are indicated by h. Residues G173, S207/E208, D239, R294, and G355/R358 lie in conserved regions 1, 3, 4, 6, and 7, respectively, of Morett and Segovia (36).

FIG. 2

Binding of MgATP and ATP to wild-type and D239 mutant forms of NtrC. The monomer concentration of NtrC was 10 μM. The amount of ATP bound to protein was measured by filter binding as described in Materials and Methods and plotted by using Kaleidograph. (A to C) Binding of MgATP to wild-type NtrC, NtrC^D239C, and NtrC^D239A, respectively; (D to F) binding of ATP to the same proteins. Since less than 1% of the ATP added was bound, [ATP]_total = [ATP]_free. All of the proteins with substitutions at D239 showed higher maximum binding of nucleotide in the presence or absence of divalent cation than did the wild-type protein (∼12% versus 4%). Given their higher apparent affinities for nucleotide, this result is commensurate with the view that low values for all proteins may be accounted for by the lability of protein-nucleotide complexes.

FIG. 3

Binding of Mn²⁺ to NtrC proteins and their isolated N-terminal domains (residues 1 to 124). Isotherms for the binding of Mn²⁺ to MBP-NtrC (178 μM monomer; a), MBP-NtrC^D239A (188 μM monomer; b), the N-terminal domain of NtrC (170 μM monomer; c, filled circles), and the N-terminal domain of NtrC^D54E (112 μM monomer; c, open circles). The amount of bound Mn²⁺ was calculated from the difference between the total amount of Mn²⁺ and the EPR measurements of Mn²⁺ that remained free in the presence of protein. The lines in panels a to c present three-point smoothing averages of the data. (d) Adjusted isotherms for MBP-NtrC (filled circles) and MBP-NtrC^D239A (open circles), corrected by subtracting nonspecific quenching from the smoothed data in panels a and b, respectively. Nonspecific quenching was estimated from the slopes of the isotherms at concentrations of free Mn²⁺ above 1 mM (see Results). The adjusted isotherms for MBP-NtrC, and MBP-NtrC^D239A were very similar, and the solid line represents a least squares fit of the combined data, which yielded a K_d of 260 μM and a stoichiometry of 1.1. Binding reactions and EPR analyses were carried out as described in Materials and Methods. [Mn²⁺]_b is [Mn²⁺]_bound, [P] is [NtrC]_total, and [Mn²⁺]_f is [Mn²⁺]_free.

FIG. 4

Titration calorimetry binding isotherm observed upon injecting MgATPγS into NtrC^3Ala protein. The titration was carried out at a dimer concentration of NtrC^3Ala of 80 μM as described in Materials and Methods. ●, experimental points; ——, fit according to the equation of Wiseman et al. (68); K_d, dissociation constant; ΔH, enthalpy change; n, stoichiometry factor. Deviations represent the fitting error of this single experiment.