SOLUTION STRUCTURE OF THE PHOSPHOCARRIER PROTEIN HPR FROM BACILLUS-SUBTILIS BY 2-DIMENSIONAL NMR-SPECTROSCOPY

The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse-detected 2D (H-1-N-15) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing N-15 assignments (reported here) as well as previously published H-1 assignments were used to identify cross-peaks that are not resolved in 2D homonuclear H-1 spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from H-1-H-2 exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X-PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 angstrom (N, C(alpha), C', O atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N(delta)1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme 1. The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40. If the betaalphabetabetaalphabeta(alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of betaalphabetabetaalphabeta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins.