Solution structure of enzyme IIA(Chitobiose) from the N,N'-diacetylchitobiose branch of the Escherichia coli phosphotransferase system.
The solution structure of trimeric Escherichia coli enzyme IIA(Chb) (34 kDa), a component of the N,N'-diacetylchitobiose/lactose branch of the phosphotransferase signal transduction system, has been determined by NMR spectroscopy. Backbone residual dipolar couplings were used to provide long range orientational restraints, and long range (|i - j| > or = 5 residues) nuclear Overhauser enhancement restraints were derived exclusively from samples in which at least one subunit was 15N/13C/2H/(Val-Leu-Ile)-methyl-protonated. Each subunit consists of a three-helix bundle. Hydrophobic residues lining helix 3 of each subunit are largely responsible for the formation of a parallel coiled-coil trimer. The active site histidines (His-89 from each subunit) are located in three symmetrically placed deep crevices located at the interface of two adjacent subunits (A and C, C and B, and B and A). Partially shielded from bulk solvent, structural modeling suggests that phosphorylated His-89 is stabilized by electrostatic interactions with the side chains of His-93 from the same subunit and Gln-91 from the adjacent subunit. Comparison with the x-ray structure of Lactobacillus lactis IIA(Lac) reveals some substantial structural differences, particularly in regard to helix 3, which exhibits a 40 degrees kink in IIA(Lac) versus a 7 degrees bend in IIA(Chb). This is associated with the presence of an unusually large (230-angstroms3) buried hydrophobic cavity at the trimer interface in IIA(Lac) that is reduced to only 45 angstroms3) in IIA(Chb).
Tang
Chun
C
Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Williams
David C
DC
Ghirlando
Rodolfo
R
Clore
G Marius
GM
D000595:Amino Acid Sequence; D001665:Binding Sites; D004187:Disaccharides / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000737:chemistry*; D012542:Scattering, Radiation; D012996:Solutions
D004187:Disaccharides; D029968:Escherichia coli Proteins; D012996:Solutions; C032438:chitobiose; D010770:Phosphotransferases