Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex.

@article{Birdsall1984MultinuclearNC,
  title={Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex.},
  author={Berry Birdsall and A. W. Bevan and Conrad Pascual and G. C. K. Roberts and James Feeney and Angela M. Gronenborn and G. Marius Clore},
  journal={Biochemistry},
  year={1984},
  volume={23 20},
  pages={
          4733-42
        }
}
The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the… 
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TLDR
In both the wild-type and mutated enzymes, exchange between the free inhibitor and the enzyme-complexed inhibitor is slow on the NMR time scale; hence, despite the considerably increased dissociation constants for binary complexes with the enzymes, the dissociation rate remains small relative to the frequency separation of the resonances.
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TLDR
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Solution structure of bound trimethoprim in its complex with Lactobacillus casei dihydrofolate reductase.
TLDR
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Conformational flexibility and protein specificity.
  • G. Roberts
  • Biology, Chemistry
    Ciba Foundation symposium
  • 1991
TLDR
The binding of substrates, inhibitors and coenzymes to the enzyme has been studied by NMR spectroscopy in conjunction with site-directed mutagenesis; changes in the chemical structure of the protein or the ligand are found to have a variety of effects on both the time-average conformation and its fluctuations.
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TLDR
The ternary complex between Lactobacillus casei dihydrofolate reductase, the coenzyme NADP+, and the antibacterial drug trimethoprim was studied by 1H and 31P NMR spectroscopy and appears to be an example of a two-state conformational equilibrium which can be "switched" by the binding of ligands of different structure.
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TLDR
The 31P NMR spectra of NADP+ and a number of its structural analogues have been obtained from their binary and ternary complexes with Lactobacillus casei dihydrofolate reductase and it is concluded that the "nicotinamide" end of the thionicotinamide and acetylpyridine coenzyme analogue binds to the enzyme quite differently from that of the natural coen enzyme NADP+.
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TLDR
It is concluded that His-64 is close to a carboxyl group in the free enzyme and that the hypoxanthine ring binds in a somewhat different orientation to the adenine ring in the NADP+ and NADPH reductase.
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Transferred nuclear Overhauser effect measurements have been made on complexes of NADP+ and thioNADP+ with Lactobacillus casei dihydrofolate reductase to provide information about the glycosidic bond
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TLDR
The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADp+ in their binary complexes with the enzyme are quite markedly different from one another.
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TLDR
The binding of trimethoprim to dihydrofolate reductase from L1210 mouse lymphoma cells has been studied by measuring the changes in chemical shift of nuclei of the ligand that accompanying binding, and it is shown that this difference arises largely from the fact that trimethobacillus casei and Escherichia coli reductases adopts different conformations when bound to mammalian and to bacteria.
Direct observation by NMR of two coexisting conformations of an enzyme–ligand complex in solution
TLDR
NMR observation by NMR of two distinct conformations of the enzyme–trimethoprim–NADP+ ternary complex in solution is reported, indicating the existence of conformational changes accompanying ligand binding and involved in inhibitor–coenzyme cooperativity.
A nuclear magnetic resonance study of nicotinamide adenine dinucleotide phosphate binding to Lactobacillus casei dihydrofolate reductase.
TLDR
The binding of NADP+ to dihydrofolate reductase and the IH spectra of the binary and ternary complexes confirm both the stoichiometry and the value of the dissociation rate constant obtained from the 13C experiments.
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TLDR
A model for the structure of the enzyme-trimethoprim complex is proposed, consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethopim.
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