An investigation of the ligand-binding site of the glutamine-binding protein of Escherichia coli using rotational-echo double-resonance NMR.
Glutamine-binding protein (GlnBP) is an essential component of the glutamine transport system in Escherichia coli. Rotational-echo double-resonance (REDOR) solid-state nuclear magnetic resonance (NMR) has been used to determine internuclear distances in the complex of GlnBP and its ligand, L-glutamine. REDOR, combined with strategically placed isotopic labels, is effective in obtaining model-independent internuclear distances and thus detailed structural information on the ligand-binding site of GlnBP. The existence of a single histidine residue (His156) in the binding site has provided an excellent probe for distance measurements between protein and ligand. REDOR distances up to 6.3 A have been observed between 13C labels in L-glutamine and 15N labels in His156. These results have unambiguously determined the ligand orientation with respect to the imidazole ring of His156, which is an important first step in refining the ligand-binding-site model of GlnBP in general. The measured distances were also used as constraints in restrained molecular dynamics calculations of the complex using the unliganded crystal structure of GlnBP as the starting point. The simulations clearly show consistency between calculated distances and those measured by REDOR.
Department of Chemistry, Washington University, St. Louis, Missouri 63130.
A W
AW
Hing
N
N
Tjandra
P F
PF
Cottam
J
J
Schaefer
C
C
Ho
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D054848:Peptaibols; D010455:Peptides; D017434:Protein Structure, Tertiary
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008024:Ligands; D054848:Peptaibols; D010455:Peptides; C037853:glutamine transport proteins; D006639:Histidine