Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two‐dimensional NMR spectroscopy

Abstract

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-I'N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing "N assignments (reported here) as well as previously published 'H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear ' H spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 'H-'H 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 A (N, C", C', 0 atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel P-sheet with two antiparallel a-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of a-helix A, with its N*1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. 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 P-turn comprised of residues 37-40. If the Pa/3Pa/3(a) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of /3aPPaP proteins that include acylphosphatase and the RNAbinding domains of the U1 snRNP A and hnRNP C proteins.Keywords: histidine-containing protein; HPr; NMR; phosphoeno1pyruvate:sugar transport system; phosphotransfer; protein structure

The solution structure of the histidine‐containing protein (HPr) from Staphylococcus aureus as determined by two‐dimensional 1H‐NMR spectroscopy

“…secondary structure motif, a four-stranded antiparallel ppleated sheet found in the NMR structure of HPr from E. coli but not in the crystal structure, was confirmed also in the NMR structures of homologeous proteins from different Gram-positive bacteria, namely in HPr from Streptococcus faecalis (Glaser, S., 1987;Hengstenberg et al, 1989;Lorenz, M., 1990), Bacillus subtilis (Wittekind et al, , 1990(Wittekind et al, , 1992 and Staphylococcus aureus (Kalbitzer et al, 1990(Kalbitzer et al, , 1991. Recently, the X-ray structures of HPr proteins from S. subtilis (Herzberg et al, 1992) and S. faecalis (Jia et al, 1993) have been solved which are in line with the NMR structures already published.…”

“…Residue 3JNl,dHz Wittekind et al (1992) for HPr from B. subtilis. Finally 12 structures were obtained which fulfilled the majority of experimental constraints and showed negative total energies.…”

Phosphorylation‐induced torsion‐angle strain in the active center of HPr, detected by NMR and restrained molecular dynamics refinement

“…This model of HPr's mode of action predicts that torsion-angle strain should be observable in other unphosphorylated HPrs as well. However, in none of the structures of HPr that have been solved since this model was proposed (Herzberg et al, 1992;Wittekind et al, 1992;Jia et al, 1993a;Kalbitzer & Hengstenberg, 1993;Liao & Herzberg, 1994;Van Nuland et al, 1994;1995) showed no strain at residue 16. As shown in Figure 1 A, the major effect of the presence of a sulphate ion in the active site of B. subtilis HPr seems to be a reorientation of the Arg 17 side chain, away from the more extended configuration in the absence of the sulphate ion and in the solution structure of E. coli HPr.…”

Phosphorylation of serine‐46 in HPr, a key regulatory protein in bacteria, results in stabilization of its solution structure

“…subtilis HPr and HPr(Ser-P) were prepared as previously described (Wittekind et al, 1989(Wittekind et al, , 1992Reizer et al, 1992) and complete phosphorylation was confirmed by electrospray mass spectrometry. NMR samples were prepared as described (Wittekind et al, 1992). Final concentrations were 1.5-2.0 mM protein, 50 mM potassium phosphate, 0.2 mM EDTA, and 2 mM sodium azide.…”

“…Structural information is available both from crystallographic (Herzberg et al, 1992) and NMR studies (Wittekind et al, 1992) of the native protein, making HPr an excellent system in which to study the effects of serine phosphorylation on the solution structure and properties of a protein regulated by this type of modification. The solution and crystal structures of HPr both clearly define an antiparallel four-stranded P-sheet flanked on one side by two long a-helices (helices A and C, Fig.…”