STRUCTURAL CONSEQUENCES OF HISTIDINE PHOSPHORYLATION - NMR CHARACTERIZATION OF THE PHOSPHOHISTIDINE FORM OF HISTIDINE-CONTAINING PROTEIN FROM BACILLUS-SUBTILIS AND ESCHERICHIA-COLI

The bacterial phosphoenolpyruvate:sugar phosphotransferase system invo lves a series of reactions in which phosphoprotein intermediates are f ormed. Histidine-containing protein (HPr) is phosphorylated on the N-d elta l position of the imidazole ring of His15 by enzyme I and acts as a phosphoryl donor to the sugar-specific enzymes IIA. The structure o f phosphorylated HPr from Bacillus subtilis, primarily, and from Esche richia coli has been studied by nuclear magnetic resonance (NMR) spect roscopy. Phosphorylation of His15 results in large downfield shifts in amide proton and nitrogen resonances for residues 16 and 17 but resul ts in only modest or no shifts in other backbone resonances. The excha nge rates of these two amide groups are decreased more than 10-fold up on phosphorylation. Analysis of the coupling constants (3)J(NH alpha) revealed no significant changes throughout the protein, indicating tha t backbone phi dihedral angles do not change detectably. (3)J(alpha be ta) and (3)J(N beta) patterns determined from P.E.COSY and HNHB spectr a, respectively, revealed a change in one side chain, that of conserve d Arg17. Analysis of NOESY spectra revealed a limited number of change s in NOEs involving protons in Ser12, His15, Arg17, and Pro18 in B. su btilis HPr. The NMR results indicate that the Arg17 side chain becomes limited in its conformational range in the phosphorylated protein, ta king on a conformation that points its guanidinium group toward the ph osphoryl group on His15. In addition, the tautomeric and ionization st ates of His15 have been determined using N-15 and P-31 NMR. At neutral pH, the imidazole is predominantly in the protonated form and the pho sphoryl group is in the dianionic form in P-His15, Altogether, the res ults indicate that phosphorylation of His15 yields only a local effect on the protein's structure. The data are consistent with a small chan ge in the disposition of the histidine side chain, allowing phosphoryl group oxygens to serve as hydrogen bond accepters for the amide proto ns of residues Ala16 and Arg17, which constitute the first two residue s of an alpha-helix. Thus, similar to many proteins that bind phosphor yl moieties noncovalently, the phosphoryl group in P-His15-HPr is situ ated to allow for a favorable electrostatic interaction at the N-termi nal end of an alpha-helix.