Three-dimensional Solution Structure of the Cytoplasmic B Domain of the Mannitol Transporter IIMannitol of the Escherichia coli Phosphotransferase System.

The solution structure of the cytoplasmic B domain of the mannitol (Mtl) transporter (IIMtl) from the mannitol branch of the Escherichia coli phosphotransferase system has been solved by multidimensional NMR spectroscopy with extensive use of residual dipolar couplings. The ordered IIBMtl domain (residues 375-471 of IIMtl) consists of a four-stranded parallel β-sheet flanked by two helices (α1 and α3) on one face and helix α2 on the opposite face with a characteristic Rossmann fold comprising two right-handed β1α1β2 and β3α2β4 motifs. The active site loop is structurally very similar to that of the eukaryotic protein tyrosine phosphatases, with the active site cysteine (Cys-384) primed in the thiolate state (pKa < 5.6) for nucleophilic attack at the phosphorylated histidine (His-554) of the IIAMtl domain through stabilization by hydrogen bonding interactions with neighboring backbone amide groups at positions i + 2/3/4 from Cys-384 and with the hydroxyl group of Ser-391 at position i + 7. Modeling of the phosphorylated state of IIBMtl suggests that the phosphoryl group can be readily stabilized by hydrogen bonding interactions with backbone amides in the i + 2/4/5/6/7 positions as well as with the hydroxyl group of Ser390 at position i + 6. Despite the absence of any significant sequence identity, the structure of IIBMtl is remarkably similar to the structures of bovine protein tyrosine phosphatase (which contains two long insertions relative to IIBMtl) and the cytoplasmic B component of enzyme IIChb, which fulfills an analogous role to IIBMtl in the N,N'-diacetylchitobiose branch of the phosphotransferase system. All three proteins utilize a cysteine residue in the nucleophilic attack of a phosphoryl group covalently bound to another protein. [ABSTRACT FROM AUTHOR]