title: Solution structure of bound trimethoprim in its complex with Lactobacillus casei dihydrofolate reductase.
abstract: Two- and three-dimensional (2D and 3D) NMR techniques have been used to assign the signals from nearly all of the protons in Lactobacillus casei dihydrofolate reductase (DHFR) (M(r) 18,300) in its 1:1 complex with the antibacterial drug trimethoprim. A sample of uniformly 15N-labeled protein was examined using 3D 15N/1H experiments [nuclear Overhauser, heteronuclear multiple quantum coherence (NOESY-HMQC) and total correlation, heteronuclear multiple quantum coherence (TOCSY-HMQC) experiments]. Twenty-two intermolecular NOEs between trimethoprim and protein protons and four intramolecular NOEs in the ligand have been detected. Some were obtained by using heteronuclear editing and 2D HMQC-NOESY experiments on complexes formed with 15N-and 13C-labeled trimethoprim molecules ([1,3-15N2,2-amino-15N]-and [7-13C,4'-methoxy-13C]trimethoprim) bound to unlabeled protein. The ligand-protein NOEs were used as distance constraints in conjunction with minimum energy and simulated annealing calculations (carried out with X-PLOR) to dock the trimethoprim ligand into dihydrofolate reductase, using as a starting structure the crystal coordinates from a related complex with a similar overall protein structure. The restrained minimum energy calculations and the simulated annealing calculations gave 83 calculated structures with distance violations of < 0.1 A. In all of these, the two aromatic rings of trimethoprim occupied essentially the same region of conformational space in the binding site (RMSD = 0.63 A). The protein residues nearest to the bound trimethoprim were found to be very similar in all of the structures and agreed well with corresponding contact residues observed in the X-ray crystal studies on trimethoprim complexes formed with Escherichia coli and chicken liver DHFRs.
authors.affiliation: Laboratory of Molecular Structure, National Institute for Medical Research, Mill Hill, London, U.K.
authors.forename: G
authors.initials: G
authors.lastname: Martorell
authors.forename: M J
authors.initials: MJ
authors.lastname: Gradwell
authors.forename: B
authors.initials: B
authors.lastname: Birdsall
authors.forename: C J
authors.initials: CJ
authors.lastname: Bauer
authors.forename: T A
authors.initials: TA
authors.lastname: Frenkiel
authors.forename: H T
authors.initials: HT
authors.lastname: Cheung
authors.forename: V I
authors.initials: VI
authors.lastname: Polshakov
authors.forename: L
authors.initials: L
authors.lastname: Kuyper
authors.forename: J
authors.initials: J
authors.lastname: Feeney
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008727:Methotrexate / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012996:Solutions; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D014295:Trimethoprim / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D012996:Solutions; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate