Structures of active site histidine mutants of IIIGlc, a major signal-transducing protein in Escherichia coli. Effects on the mechanisms of regulation and phosphoryl transfer.
IIIGlc (also called IIAGlc), a major signal-transducing protein in Escherichia coli, is also a phosphorylcarrier in glucose uptake. The crystal and NMR structures of IIIGlc show that His90, the phosphoryl acceptor, adjoins His75 in the active site. Glutamine was substituted for His-, giving H75QIIIGlc and H90QIIIGlc, respectively (Presper, K. A., Wong, C.-Y., Liu, L., Meadow, N. D., and Roseman, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4052-4055), but the mutants showed unexpected properties. H90QIIIGlc loses regulatory functions of IIIGlc, and the phosphoryltransfer rates between HPr/H75QIIIGlc are 200-fold less than HPr/IIIGlc (Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33440-33445). X-ray crystallography, differential scanning calorimetry, and NMR have now been used to determine the structures of the mutants (phospho-H75QIIIGlc was studied by NMR). The three methods gave completely consistent results. Except for the His to Gln substitutions, the only significant structural changes were in a few hydrogen bonds. H90QIIIGlc contains two structured water molecules (to Gln90), which could explain its inability to regulate glycerol kinase. Phospho-IIIGlc contains a chymotrypsin-like, hydrogen bond network (Thr73-His75-O--phosphoryl), whereas phospho-H75QIIIGlc contains only one bond (Gln75-O--phosphoryl). Hydrogen bonds play an essential role in a proposed mechanism for the phosphoryltransfer reaction.
Pelton
J G
JG
Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Torchia
D A
DA
Remington
S J
SJ
Murphy
K P
KP
Meadow
N D
ND
Roseman
S
S
D001665:Binding Sites; D002151:Calorimetry; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D006639:Histidine; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D010766:Phosphorylation
D029968:Escherichia coli Proteins; C032242:crr protein, E coli; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System