Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Jun;85(11):3787–3791. doi: 10.1073/pnas.85.11.3787

Dynamics of trimethoprim bound to dihydrofolate reductase.

M S Searle 1, M J Forster 1, B Birdsall 1, G C Roberts 1, J Feeney 1, H T Cheung 1, I Kompis 1, A J Geddes 1
PMCID: PMC280304  PMID: 3131763

Abstract

The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, we report the use of 1H and 13C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its "target," dihydrofolate reductase. 13C relaxation measurements reveal dihedral angle changes of +/- 25 degrees to +/- 35 degrees on the subnanosecond time scale, while 13C line-shape analysis demonstrates dihedral angle changes of at least +/- 65 degrees on the millisecond time scale. 1H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.

Full text

PDF

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baccanari D. P., Daluge S., King R. W. Inhibition of dihydrofolate reductase: effect of reduced nicotinamide adenine dinucleotide phosphate on the selectivity and affinity of diaminobenzylpyrimidines. Biochemistry. 1982 Sep 28;21(20):5068–5075. doi: 10.1021/bi00263a034. [DOI] [PubMed] [Google Scholar]
  2. Baker D. J., Beddell C. R., Champness J. N., Goodford P. J., Norrington F. E., Smith D. R., Stammers D. K. The binding of trimethoprim to bacterial dihydrofolate reductase. FEBS Lett. 1981 Apr 6;126(1):49–52. doi: 10.1016/0014-5793(81)81030-7. [DOI] [PubMed] [Google Scholar]
  3. Bevan A. W., Roberts G. C., Feeney J., Kuyper L. 1H and 15N NMR studies of protonation and hydrogen-bonding in the binding of trimethoprim to dihydrofolate reductase. Eur Biophys J. 1985;11(4):211–218. doi: 10.1007/BF00261997. [DOI] [PubMed] [Google Scholar]
  4. Birdsall B., Burgen A. S., Roberts G. C. Binding of coenzyme analogues to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes. Biochemistry. 1980 Aug 5;19(16):3723–3731. doi: 10.1021/bi00557a013. [DOI] [PubMed] [Google Scholar]
  5. Birdsall B., Burgen A. S., Roberts G. C. Effects of coenzyme analogues on the binding of p-aminobenzoyl-L-glutamate and 2,4-diaminopyrimidine to Lactobacillus casei dihydrofolate reductase. Biochemistry. 1980 Aug 5;19(16):3732–3737. doi: 10.1021/bi00557a014. [DOI] [PubMed] [Google Scholar]
  6. Bolin J. T., Filman D. J., Matthews D. A., Hamlin R. C., Kraut J. Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. I. General features and binding of methotrexate. J Biol Chem. 1982 Nov 25;257(22):13650–13662. [PubMed] [Google Scholar]
  7. Burgen A. S., Roberts G. C., Feeney J. Binding of flexible ligands to macromolecules. Nature. 1975 Feb 27;253(5494):753–755. doi: 10.1038/253753a0. [DOI] [PubMed] [Google Scholar]
  8. Campbell I. D., Dobson C. M., Williams R. J. Proton magnetic resonance studies of the tyrosine residues of hen lysozyme-assignment and detection of conformational mobility. Proc R Soc Lond B Biol Sci. 1975 Jun 17;189(1097):503–509. doi: 10.1098/rspb.1975.0070. [DOI] [PubMed] [Google Scholar]
  9. Case D. A., Karplus M. Dynamics of ligand binding to heme proteins. J Mol Biol. 1979 Aug 15;132(3):343–368. doi: 10.1016/0022-2836(79)90265-1. [DOI] [PubMed] [Google Scholar]
  10. Cayley P. J., Albrand J. P., Feeney J., Roberts G. C., Piper E. A., Burgen A. S. Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase. Biochemistry. 1979 Sep 4;18(18):3886–3895. doi: 10.1021/bi00585a008. [DOI] [PubMed] [Google Scholar]
  11. Cheung H. T., Searle M. S., Feeney J., Birdsall B., Roberts G. C., Kompis I., Hammond S. J. Trimethoprim binding to Lactobacillus casei dihydrofolate reductase: a 13C NMR study using selectively 13C-enriched trimethoprim. Biochemistry. 1986 Apr 22;25(8):1925–1931. doi: 10.1021/bi00356a014. [DOI] [PubMed] [Google Scholar]
  12. Clore G. M., Gronenborn A. M., Birdsall B., Feeney J., Roberts G. C. 19F-n.m.r. studies of 3',5'-difluoromethotrexate binding to Lactobacillus casei dihydrofolate reductase. Molecular motion and coenzyme-induced conformational changes. Biochem J. 1984 Feb 1;217(3):659–666. doi: 10.1042/bj2170659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cocco L., Roth B., Temple C., Jr, Montgomery J. A., London R. E., Blakley R. L. Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase. Arch Biochem Biophys. 1983 Oct 15;226(2):567–577. doi: 10.1016/0003-9861(83)90326-0. [DOI] [PubMed] [Google Scholar]
  14. Dann J. G., Ostler G., Bjur R. A., King R. W., Scudder P., Turner P. C., Roberts G. C., Burgen A. S. Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei. Biochem J. 1976 Sep 1;157(3):559–571. doi: 10.1042/bj1570559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Englander S. W., Kallenbach N. R. Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q Rev Biophys. 1983 Nov;16(4):521–655. doi: 10.1017/s0033583500005217. [DOI] [PubMed] [Google Scholar]
  16. Feeney J., Birdsall B., Albrand J. P., Roberts G. C., Burgen A. S., Charlton P. A., Young D. W. Hydrogen-1 nuclear magnetic resonance study of the complexes of two diastereoisomers of folinic acid with dihydrofolate reductase. Biochemistry. 1981 Mar 31;20(7):1837–1842. doi: 10.1021/bi00510a019. [DOI] [PubMed] [Google Scholar]
  17. Frauenfelder H., Petsko G. A., Tsernoglou D. Temperature-dependent X-ray diffraction as a probe of protein structural dynamics. Nature. 1979 Aug 16;280(5723):558–563. doi: 10.1038/280558a0. [DOI] [PubMed] [Google Scholar]
  18. Hammond S. J., Birdsall B., Searle M. S., Roberts G. C., Feeney J. Dihydrofolate reductase. 1H resonance assignments and coenzyme-induced conformational changes. J Mol Biol. 1986 Mar 5;188(1):81–97. doi: 10.1016/0022-2836(86)90483-3. [DOI] [PubMed] [Google Scholar]
  19. Huber R., Bennett W. S., Jr Functional significance of flexibility in proteins. Biopolymers. 1983 Jan;22(1):261–279. doi: 10.1002/bip.360220136. [DOI] [PubMed] [Google Scholar]
  20. Jardetzky O., Akasaka K., Vogel D., Morris S., Holmes K. C. Unusual segmental flexibility in a region of tobacco mosaic virus coat protein. Nature. 1978 Jun 15;273(5663):564–566. doi: 10.1038/273564a0. [DOI] [PubMed] [Google Scholar]
  21. Karplus M., McCammon J. A. Dynamics of proteins: elements and function. Annu Rev Biochem. 1983;52:263–300. doi: 10.1146/annurev.bi.52.070183.001403. [DOI] [PubMed] [Google Scholar]
  22. Koetzle T. F., Williams G. J. The crystal and molecular structure of the antifolate drug trimethoprim (2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine). A neutron diffraction study. J Am Chem Soc. 1976 Apr 14;98(8):2074–2078. doi: 10.1021/ja00424a009. [DOI] [PubMed] [Google Scholar]
  23. Levitt M., Sander C., Stern P. S. Protein normal-mode dynamics: trypsin inhibitor, crambin, ribonuclease and lysozyme. J Mol Biol. 1985 Feb 5;181(3):423–447. doi: 10.1016/0022-2836(85)90230-x. [DOI] [PubMed] [Google Scholar]
  24. Matthews D. A., Bolin J. T., Burridge J. M., Filman D. J., Volz K. W., Kaufman B. T., Beddell C. R., Champness J. N., Stammers D. K., Kraut J. Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim. J Biol Chem. 1985 Jan 10;260(1):381–391. [PubMed] [Google Scholar]
  25. Northrup S. H., Pear M. R., Lee C. Y., McCammon J. A., Karplus M. Dynamical theory of activated processes in globular proteins. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4035–4039. doi: 10.1073/pnas.79.13.4035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nowak T., Mildvan A. S. Nuclear magnetic resonance studies of selectively hindered internal motion of substrate analogs at the active site of pyruvate kinase. Biochemistry. 1972 Jul 18;11(15):2813–2818. doi: 10.1021/bi00765a013. [DOI] [PubMed] [Google Scholar]
  27. Perham R. N., Duckworth H. W., Roberts G. C. Mobility of polypeptide chain in the pyruvate dehydrogenase complex revealed by proton NMR. Nature. 1981 Jul 30;292(5822):474–477. doi: 10.1038/292474a0. [DOI] [PubMed] [Google Scholar]
  28. Roberts G. C., Feeney J., Burgen A. S., Daluge S. The charge state of trimethoprim bound to Lactobacillus casei dihydrofolate reductase. FEBS Lett. 1981 Aug 17;131(1):85–88. doi: 10.1016/0014-5793(81)80893-9. [DOI] [PubMed] [Google Scholar]
  29. Thomas D. D. Large-scale rotational motions of proteins detected by electron paramagnetic resonance and fluorescence. Biophys J. 1978 Nov;24(2):439–462. doi: 10.1016/S0006-3495(78)85394-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wagner G. Characterization of the distribution of internal motions in the basic pancreatic trypsin inhibitor using a large number of internal NMR probes. Q Rev Biophys. 1983 Feb;16(1):1–57. doi: 10.1017/s0033583500004911. [DOI] [PubMed] [Google Scholar]
  31. Woody R. W., Clark D. C., Roberts G. C., Martin S. R., Bayley P. M. Molecular flexibility in microtubule proteins: proton nuclear magnetic resonance characterization. Biochemistry. 1983 Apr 26;22(9):2186–2192. doi: 10.1021/bi00278a020. [DOI] [PubMed] [Google Scholar]
  32. Wüthrich K., Wagner G. NMR investigations of the dynamics of the aromatic amino acid residues in the basic pancreatic trypsin inhibitor. FEBS Lett. 1975 Feb 1;50(2):265–268. doi: 10.1016/0014-5793(75)80504-7. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES