Regulation of the pho regulon of Escherichia coli K-12. Cloning of the regulatory genes phoB and phoR and identification of their gene products

We report here results of crystallographic studies at 3.0 Å resolution of complexes of phosphate ligands with aspartate carbamoyltransferase from Escherichia coli. Specifically, we interpret the binding of CTP, ATP, 5-bromo-CTP, 8-bromo-GTP. formycin A 5′-triphosphate. 3,N⁶-etheno-ATP. phosphate/carbamoyl-d.l-aspartate and pyrophosphate to the catalytic and regulatory chains of the enzyme.We observed two modes of binding of ligands to the phosphate crevice of the catalytic chain. Pyrophosphate and phosphate penetrate deeply into the cleft between the two domains of a catalytic monomer. In contrast. ATP, CTP. formycin A 5′-triphosphate and 3,N⁶-etheno-ATP bind to an exposed region of this cleft through their β and γ phosphates. Although the β and γ phosphates of 8-bromo-GTP bind to the same region as do the non-brominated nucleotides. 8-bromo-GTP interacts with the protein through all three of its phosphates and its ribose.Ser52, Arg54. Thr55, Arg105, His134. Gln137 and Arg167 are residues of the catalytic chain near density corresponding to phosphate ligands. The interactions of phosphate ligands are consistent with results of nuclear magnetic resonance, kinetics and equilibrium binding studies.Nucleoside triphosphates also bind to the regulatory chain in two modes. ATP and CTP bind in similar conformations to nearly the same site of the allosteric domain. The effector 8-bromo-GTP interacts at a location that does not overlap with the ATP-CTP site. The phosphates are in an extended conformation for all effectors. Furthermore, ATP. 5-bromo-CTP and 8-bromo-GTP bind to the protein in the anti conformation.Interactions of ATP and CTP with the protein are essentially consistent with the proposals put forward by London &; Schmidt (1972). We suggest, however, a modification of the London &; Schmidt model on the basis of our results with 8-bromo-GTP. In addition, we propose that the allosteric binding sites of nucleoside triphosphates are coupled to each other through the N-terminal segments of monomers of a regulatory dimer.