EurekaMag.com logo
+ Site Statistics
References:
53,623,987
Abstracts:
29,492,080
+ Search Articles
+ Subscribe to Site Feeds
EurekaMag Most Shared ContentMost Shared
EurekaMag PDF Full Text ContentPDF Full Text
+ PDF Full Text
Request PDF Full TextRequest PDF Full Text
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter
Follow on LinkedInFollow on LinkedIn

+ Translate

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



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



Biochemistry 23(20): 4733-4742



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 environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.

(PDF same-day service: $19.90)

Accession: 018017280

Download citation: RISBibTeXText

PMID: 6437442

DOI: 10.1021/bi00315a032



Related references

Multinuclear nmr characterization of 2 coexisting conformational states of the lactobacillus casei dihydrofolate reductase trimethoprim nadp complex. Biochemistry 23(20): 4733-4742, 1984

Solution structure of bound trimethoprim in its complex with Lactobacillus casei dihydrofolate reductase. Biochemistry 33(41): 12416-12426, 1994

NMR-based solution structure of the complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADPH. Journal of Biomolecular Nmr 24(1): 67-70, 2002

Characterization of rates of ring-flipping in trimethoprim in its ternary complexes with Lactobacillus casei dihydrofolate reductase and coenzyme analogues. Biochemistry 38(48): 15962-9, 2000

Characterization of rates of ring-flipping in trimethoprim in its ternary complexes with Lactobacillus casei dihydrofolate reductase and coenzyme analogues. Biochemistry (American Chemical Society) 38(48): 962-9, 1999

Trimethoprim binding to Lactobacillus casei dihydrofolate reductase: a 13C NMR study using selectively 13C-enriched trimethoprim. Biochemistry 25(8): 1925-1931, 1986

1H and 31P NMR characterization of two conformations of the trimethoprim-NADP+-dihydrofolate reductase complex. Molecular Pharmacology 20(1): 145-153, 1981

31P-MNR studies of NADPH, NADP+ and the complex of NADPH and Methotrexate with Lactobacillus casei dihydrofolate reductase in the solid state. European journal of biochemistry 226(1): 211-218, 1994

Hydrogen-1, carbon-13, and phosphorus-31 nuclear magnetic resonance studies of the dihydrofolate reductase-nicotinamide adenine dinucleotide phosphate-folate complex: characterization of three coexisting conformational states. Biochemistry 21(23): 5831-5838, 1982

Proton nmr carbon 13 nmr and phosphorus 31 nmr studies of the di hydro folate reductase nadp folate complex characterization of 3 coexisting conformational states. Biochemistry 21(23): 5831-5838, 1982

The charge state of trimethoprim bound to Lactobacillus casei dihydrofolate reductase. Febs Letters 131(1): 85-88, 1981

Trimethoprim binding to lactobacillus casei dihydrofolate reductase a carbon 13 nmr study using selectively carbon 13 enriched trimethoprim. Biochemistry 25(8): 1925-1931, 1986

Immunochemical evidence for extensive ligand-induced conformational changes in Lactobacillus casei dihydrofolate reductase. Journal of Biological Chemistry 259(2): 1082-1085, 1984