NMR structure of cysteinyl-phosphorylated enzyme IIB of the N,N '-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase systemof Escherichia coli

The determination by NMR of the solution structure of the phosphorylated en zyme IIB (P-IIBChb) Of the N,N'-diacetylchitobiose-specific phosphoenolpyru vate-dependent phosphotransferase system of Escherichia coil is presented. Most of the backbone and side-chain resonances were assigned using a variet y of mostly heteronuclear NMR experiments. The remaining resonances were as signed with the help of the structure calculations. NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addit ion, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous rest raints into new ambiguous restraints, an error function was constructed tha t was less sensitive to information loss caused by assignment uncertainties . The final set of structures had a pairwise rmsd of 0.59 Angstrom and 1.16 Angstrom for the heavy atoms of the backbone and sidechains, respectively. Comparing the P-IIBChb solution structure with the previously determined NM R and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are Limited to the active-si te region. The phosphoryl group at the active-site cysteine residue is surr ounded by a loop formed by residues 10 through 16. NOE and chemical shift d ata suggest that the phosphoryl group makes hydrogen bonds with the backbon e amide protons of residues 12 and 15. The binding mode of the phosphoryl g roup is very similar to that of the protein tyrosine phosphatases. The diff erences observed are in accordance with the presumption that IIBChb has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cyste ine SH proton and the solvent via the hydroxyl group of Thr16. (C) 2001 Aca demic Press.