Visualization of the phosphorylated active site loop of the cytoplasmic B domain of the mannitol transporter II(Mannitol) of the Escherichia coli phosphotransferase system by NMR spectroscopy and residual dipolar couplings.
The solution structure of a stably phosphorylated form of the cytoplasmic B domain of the mannitol-specific transporter (IIB(Mtl)) of the Escherichia coli phosphotransferase system, containing a mutation of the active site Cys384 to Ser, has been solved by NMR. The strategy employed relies principally on backbone residual dipolar couplings recorded in three different alignment media, supplemented by nuclear Overhauser enhancement data and torsion angle restraints related specifically to the active site loop (residues 383-393). As judged from the dipolar coupling data, the remainder of the structure is unchanged upon phosphorylation within the errors of the coordinates of the previously determined solution structure of unphosphorylated wild-type IIB(Mtl). Thus, only the active site loop was refined. Phosphorylation results in a backbone atomic rms shift of approximately 0.7 angstroms in the active site loop. The resulting conformation is less than 0.5 angstroms away from the equivalent P-loop in both the low and high molecular mass eukaryotic tyrosine phosphatases. 3J(NP) coupling constant measurements using quantitative J-correlation spectroscopy provide a direct demonstration of a hydrogen bond between the phosphoryl group and the backbone amide of Ser391 at position i + 7 from phospho-Ser384, with an approximately linear P-O-H(N) bond angle. The structure also reveals additional hydrogen bonding interactions involving the backbone amides of residues at positions i + 4 and i + 5, and the hydroxyl groups of two serine residues at positions i + 6 and i + 7 that stabilize the phosphoryl group.
Suh
Jeong-Yong
JY
Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA.
Tang
Chun
C
Cai
Mengli
M
Clore
G Marius
GM
D001665:Binding Sites; D003593:Cytoplasm; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D008353:Mannitol; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary
D029968:Escherichia coli Proteins; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C037992:phosphoenolpyruvate-mannose phosphotransferase