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A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site

Nature Structural Biology volume 2, pages 1018–1025 (1995)Cite this article

Abstract

Bacteria expressing R67-plasmid encoded dihydrofolate reductase (R67 DHFR) exhibit high-level resistance to the antibiotic trimethoprim. Native R67 DHFR is a 34,000 Mr homotetramer which exists in equilibrium with an inactive dimeric form. The structure of native R67 DHFR has now been solved at 1.7 Å resolution and is unrelated to that of chromosomal DHFR. Homotetrameric R67 DHFR has an unusual pore, 25 Å in length, passing through the middle of the molecule. Two folate molecules bind asymmetrically within the pore indicating that the enzyme's active site consists of symmetry related binding surfaces from all four identical units.

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References

  1. Kraut, J. & Matthews, D.A. in Active sites of Enzymes (eds Jurnak, F.A. & McPherson, A.) 3, 1–71 (John Wiley & sons, New York, 1987).

    Google Scholar 

  2. Fleming, M.P., Datta, N. & Gruneberg, R.N. Trimethoprim resistance determined by R factors. Br. med. J. 1, 726–728 (1972).

    Article  CAS  Google Scholar 

  3. Pattishall, K.H. et al. Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups. J. biol. Chem. 252, 2319–2323 (1977).

    CAS  PubMed  Google Scholar 

  4. Smith, S.L. et al. R plasmid dihydrofolate reductase with subunit structure. J. biol. Chem. 254, 6222–6225 (1979).

    CAS  PubMed  Google Scholar 

  5. Stone, D. & Smith, S.L. The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67. J. biol. Chem. 254, 10857–10861 (1979).

    CAS  PubMed  Google Scholar 

  6. Nichols, R. et al. Titration of histidine 62 in R67 dihydrofolate reductase is linked to a tetramer ⟺ two-dimer equilibrium. Biochemistry 32, 1695–1706 (1993).

    Article  CAS  Google Scholar 

  7. Matthews, D.A. et al. Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67. Biochemistry 25, 4194–4204 (1986).

    Article  CAS  Google Scholar 

  8. Reece, L.J., Nichols, R., Ogden, R.C. & Howell, E.E. Construction of a synthetic gene for an R-plasmid encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein. Biochemistry 30, 10895–10904 (1991).

    Article  CAS  Google Scholar 

  9. Janin, J., Miller, S. & Chothia, C. Surface, subunit interfaces and interior of oligomeric proteins. J. molec. Biol. 204, 155–164 (1988).

    Article  CAS  Google Scholar 

  10. Braig, K. et al. The crystal structure of the bacterial chaperonin GroEL at 2.8 Å. Nature 371, 578–586 (1994).

    Article  CAS  Google Scholar 

  11. Bennett, M.J., Choe, S. & Eisenberg, D. Domain swapping: entangling alliances between proteins. Proc. natn. Acad. Sci. U.S.A. 91, 3127–3131 (1994).

    Article  CAS  Google Scholar 

  12. Matthews, B.W. & Bernhard, S.A. Structure and symmetry of oligomeric enzymes. A. Rev. Biophys. Bioengng. 2, 257–317 (1973).

    Article  CAS  Google Scholar 

  13. Navia, M.A. et al. Three-dimensional structure of aspartyl protease from human immuno deficiency virus HIV-1. Nature 337, 615–620 (1989).

    Article  CAS  Google Scholar 

  14. Holland, J.C. et al. in Proc 10th Intl. symp. chem. biol. pteridines (eds Ayling, J. E. et al.) 493–498 (Plenum, New York, 1993).

    Google Scholar 

  15. Brito, R.M.M. et al. Characterization and stereochemistry of cofactor oxidation by a type II dihydrofolate reductase. Biochemistry 29, 9825–9831 (1990).

    Article  CAS  Google Scholar 

  16. Morrison, J.F. & Sneddon, M.K. in Chemistry and Biology of Pteridines (eds Curtius, H.-Ch., Ghisla, S. & Bau, N.) 728–733 (Walter de Gruyter, Berlin, 1990).

    Google Scholar 

  17. Bystroff, C., Oatley, S.J. & Kraut, J. Crystal structures of Escherichia coli dihydrofolate reductase: The NADP+ holoenzyme and the folate. NADP+ ternary complex. Substrate binding and a model for the transition state. Biochemistry 29, 3263–3277 (1990).

    Article  CAS  Google Scholar 

  18. Brito, R.M.M., Rudolph, F.B. & Rosevear, P.R. Conformation of NADP+ bound to a type II dihydrofolate reductase. Biochemistry 30, 1461–1469 (1991).

    Article  CAS  Google Scholar 

  19. Smith, S.L. & Burchall, J. α-Pyridine nucleotides as substrates for a plasmid-specified dihydrofolate reductase. Proc. natn. Acad. Sci. U.S.A. 80, 4619–4623 (1983).

    Article  CAS  Google Scholar 

  20. Borchert, T.V., Mathieu, M. & Zeelen, J.P. The crystal structure of human Csk SH3: Structural diversity near the RT-Src and n-Src loop. FEBS Lett. 341, 79–85 (1994).

    Article  CAS  Google Scholar 

  21. Kohda, D. et al. Solution structure and ligand-binding site of the carboxy-terminal SH3 domain of GRB2. Structure 2, 1029–1040 (1994).

    Article  CAS  Google Scholar 

  22. Musacchio, A., Saraste, M. & Wilmanns, M. High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides. Nature struct. Biol. 1, 546–551 (1994).

    Article  CAS  Google Scholar 

  23. Wilson, K.P. et al. Escherichia coli biotin holoenzyme synthetase/ bio repressor crystal structure delineates the biotin- and DNA-binding domains. Proc. natn. Acad. Sci. U.S.A. 89, 9257–9261 (1992).

    Article  CAS  Google Scholar 

  24. Falzone, C.J. et al. Three-dimensional solution structure of PsaE from the cyanobacterium synechococcus sp. strain PCC 7002, a photosystem I protein that shows structural homology with SH3 domains. Biochemistry 33, 6052–6062 (1994).

    Article  CAS  Google Scholar 

  25. Baumann, H. et al. Solution structure and DNA-binding properties of a thermostable protein from the archaeon sulfolobus solfataricus. Nature struct. Biol. 1, 808–819 (1994).

    Article  CAS  Google Scholar 

  26. Jencks, W.P. Catalysis in Chem. and Enzym., (McGraw-Hill, New York; 1975).

    Google Scholar 

  27. Matthews, B.W. Solvent content of protein crystals. J. molec. Biol. 33, 491–497 (1968).

    Article  CAS  Google Scholar 

  28. Hamlin, R. Multiwire area X-ray diffractometers. Meths Enzym. 114A, 416–452 (1985).

    Article  Google Scholar 

  29. Howard, A.J., Nielsen, C., Xuong, N.-h. Software for a diffractometer with multiwire area detector. Meths. Enzym. 114, 452–472 (1985).

    Article  CAS  Google Scholar 

  30. Eleanor, D. et al. A programme system from Oxford, UK.

  31. Terwilliger, T.C. & Eisenberg, D. Unbiased three-dimensional refinement of heavy-atom parameters by correlation of origin-removed patterson functions. Acta crystallogr. A39, 813–817 (1983).

    Article  CAS  Google Scholar 

  32. Wang, B.-C. Resolution of phase ambiguity in macromolecular crystallography. Meths Enzym. 115B, 90–112 (1985).

    Article  Google Scholar 

  33. Cambillau, C. & Horjales, E. TOM: a FRODO subpackage for protein-ligand fitting with interactive energy minimization. J. molec. Graph. 5, 174–177 (1987).

    Article  CAS  Google Scholar 

  34. Brunger, A.T. X-PLOR vers. 3.1 Manual: A system for X-ray Crystallography & NMR (1992). Yale University, New Haven, CT 06511, U.S.A.

    Google Scholar 

  35. Tronrud, D.E., Ten Eyck, L.F. & Matthews, B.W. An efficient general-purpose least-squares refinement program for macromolecular structures. Acta crystallogr. A43, 489–501 (1987).

    Article  CAS  Google Scholar 

  36. Laskowski, R.A., MacArthur, M.W., Moss, D.S., & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  37. Ramachandran, G.N. & Sasisekharan, V. Conformation of polypeptides and proteins. Adv. prot. Chem. 23, 283–438 (1968).

    CAS  Google Scholar 

  38. Luzzati, P.V. Traitement statistique des erreurs dans la determination des structures cristallines. Acta crystallogr. 5, 802–810 (1952).

    Article  Google Scholar 

  39. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  40. Insight II User Guide, Vers. 2.3.9 (1994), Biosym Technologies, Inc., San Diego.

  41. Connolly, M.L. Analytical molecular surface calculation. J. appl. Crystallogr. 16, 548–558 (1983).

    Article  CAS  Google Scholar 

  42. Bolin, J.T. et al. Crystal structures of Escherichia coli and Lactobacillus casei dihydro-folate reductase refined a 1.7 Å resolution. I. General features and binding of methotrexate. J. biol. Chem. 257, 13650–13662 (1982).

    CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, 0317, University of California, 9500 Gilman Drive, San Diego, La Jolla, California, 92093, USA

    Narendra Narayana

  2. Agouron Pharmaceuticals, Incorporated, 3565 General Atomics Court, San Diego, California, 92121, USA

    David A. Matthews

  3. Department of Biochemistry, University of Tennessee, Knoxville, Tennessee, 37996-0840, USA

    Elizabeth E. Howell

  4. Departments of Biology, Physics and Chemistry and Biochemistry, 0317, University of California, San Diego, 9500 Gilman Drive, La Jolla, California, 92093, USA

    Nguyen-huu Xuong

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  2. David A. Matthews
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  3. Elizabeth E. Howell
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  4. Nguyen-huu Xuong
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Narayana, N., Matthews, D., Howell, E. et al. A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site. Nat Struct Mol Biol 2, 1018–1025 (1995). https://doi.org/10.1038/nsb1195-1018

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