title: Investigation of ligand binding and protein dynamics in Bacillus subtilis chorismate mutase by transverse relaxation optimized spectroscopy-nuclear magnetic resonance.
abstract: The structural and dynamical consequences of ligand binding to a monofunctional chorismate mutase from Bacillus subtilis have been investigated by solution NMR spectroscopy. TROSY methods were employed to assign 98% of the backbone (1)H(N), (1)H(alpha), (15)N, (13)C', and (13)C(alpha) resonances as well as 86% of the side chain (13)C resonances of the 44 kDa trimeric enzyme at 20 degrees C. This information was used to map chemical shift perturbations and changes in intramolecular mobility caused by binding of prephenate or a transition state analogue to the X-ray structure. Model-free interpretation of backbone dynamics for the free enzyme and its complexes based on (15)N relaxation data measured at 600 and 900 MHz showed significant structural consolidation of the protein in the presence of a bound ligand. In agreement with earlier structural and biochemical studies, substantial ordering of 10 otherwise highly flexible residues at the C-terminus is particularly notable. The observed changes suggest direct contact between this protein segment and the bound ligand, providing support for the proposal that the C-terminus can serve as a lid for the active site, limiting diffusion into and out of the pocket and possibly imposing conformational control over substrate once bound. Other regions of the protein that experience substantial ligand-induced changes also border the active site or lie along the subunit interfaces, indicating that the enzyme adapts dynamically to ligands by a sort of induced fit mechanism. It is believed that the mutase-catalyzed chorismate-to-prephenate rearrangement is partially encounter controlled, and backbone motions on the millisecond time scale, as seen here, may contribute to the reaction barrier.
authors.affiliation: Department of Chemistry and Applied Bioscience, Swiss Federal Institute of Technology, ETH Hönggerberg, 8093 Zürich, Switzerland.
authors.forename: Alexander
authors.initials: A
authors.lastname: Eletsky
authors.forename: Alexander
authors.initials: A
authors.lastname: Kienhöfer
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
authors.forename: Konstantin
authors.initials: K
authors.lastname: Pervushin
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D002826:Chorismate Mutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D003509:Cyclohexanecarboxylic Acids / Q000737:chemistry / Q000378:metabolism; D053138:Cyclohexenes; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011522:Protons; D012996:Solutions; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D008024:Ligands; D009587:Nitrogen Isotopes; D011522:Protons; D012996:Solutions; D002826:Chorismate Mutase; C005550:prephenic acid