Polypeptide backbone resonance assignments and secondary structure of Bacillus subtilis enzyme IIIglc determined by two-dimensional and three-dimensional heteronuclear NMR spectroscopy.
The enzyme IIIglc-like domain of Bacillus subtilis IIglc (IIIglc, 162 residues, 17.4 kDa) has been cloned and overexpressed in Escherichia coli. Sequence-specific assignment of the backbone 1H and 15N resonances has been carried out with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional (3D) NMR spectroscopy. Amide proton solvent exchange rate constants have been determined from a series of 1H-15N heteronuclear single-quantum coherence (HSQC) spectra acquired following dissolution of the protein in D2O. Major structural features of IIIglc have been inferred from the pattern of short-, medium- and long-range NOEs in 3D heteronuclear 1H nuclear Overhauser effect 1H-15N multiple-quantum coherence (3D NOESY-HMQC) spectra, together with the exchange rate constants. IIIglc contains three antiparallel beta-sheets comprised of eight, three, and two beta-strands. In addition, five beta-bulges were identified. No evidence of regular helical structure was found. The N-terminal 15 residues of the protein appear disordered, which is consistent with their being part of the Q-linker that connects the C-terminal enzyme IIIglc-like domain to the membrane-bound IIglc domain. Significantly, two histidine residues, His 68 and His 83, which are important for phosphotransferase function, are found from NOE measurements to be in close proximity at the ends of adjacent strands in the major beta-sheet.
Fairbrother
W J
WJ
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Cavanagh
J
J
Dyson
H J
HJ
Palmer
A G
AG
Sutrina
S L
SL
Reizer
J
J
Saier
M H
MH
Wright
P E
PE
D000577:Amides; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation
D000577:Amides; D010455:Peptides; D010750:Phosphoproteins; C033623:factor III(Glc); D010731:Phosphoenolpyruvate Sugar Phosphotransferase System