A productive NADP+ binding mode of ferredoxin–NADP+ reductase revealed by protein engineering and crystallographic studies

Article metrics

Abstract

The flavoenzyme ferredoxin–NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin–nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of ~30°, with the C4 atom 3 Å from the flavin N5 atom.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Spectroscopic characterization of wild type, Y308S and Y308W pea FNRs.
Figure 2: NADP+ binding mode in Y308S–NADP+ complex.
Figure 3: NADP(H) binding to pea FNR mutants.

References

  1. 1

    Arakaki, A.K., Ceccarelli, E.A. & Carrillo, N. FASEB J. 11, 133– 140 (1997).

  2. 2

    Karplus, P.A., Daniels, M.J. & Herriott, J.R. Science 251, 60– 66 (1991).

  3. 3

    Bruns, C.M. & Karplus, P.A. J. Mol. Biol. 247, 125–145 (1995).

  4. 4

    Serre, L. et al. J. Mol. Biol. 263, 20– 39 (1996).

  5. 5

    Correll, C.C., Ludwig, M.L., Bruns, C.M. & Karplus, P.A. Protein Sci. 2, 2112–2133 (1993).

  6. 6

    Ingelman, M., Bianchi, V. & Eklund, H. J. Mol. Biol. 268, 147– 157 (1997).

  7. 7

    Nishida, H. et al. Biochemistry 34, 2763– 2767 (1995).

  8. 8

    Lu, G., Campbell, W.H., Schneider, G. & Lindqvist, Y. Structure (London) 2, 809–821 (1994).

  9. 9

    Correll, C.C., Batie, C.J., Ballou, D.P. & Ludwig, M.L. Science 258, 1604–1610 ( 1992).

  10. 10

    Wang, M. et al. Proc. Natl. Acad. Sci. USA 94, 8411– 8416 (1997).

  11. 11

    Pai, E., Karplus, P.A. & Schulz, G.E. Biochemistry 27, 4465– 4474 (1988).

  12. 12

    Karplus, P.A. & Schulz, G.E. J. Mol. Biol. 210, 163–180 (1989).

  13. 13

    Stehle, T., Claiborne, A. & Schulz, G.E. Eur. J. Biochem. 211, 221– 226 (1993).

  14. 14

    Li, R., Bianchet, M.A., Talalay, P. & Amzel, L.M. Proc. Natl. Acad. Sci. USA 92, 8846– 8850 (1995).

  15. 15

    Orellano, E.G., Calcaterra, N.B., Carrillo, N. & Ceccarelli, E.A. J. Biol. Chem. 268, 19267–19273 (1993).

  16. 16

    Calcaterra,N.B. et al. Biochemistry 34, 12842– 12848 (1995).

  17. 17

    Aliverti, A., Gadda, G., Ronchi, S., and Zanetti, G. Eur. J. Biochem 198, 21–24 ( 1991).

  18. 18

    Aliverti, A., Lubberstedt, T., Zanetti, G., Herrmann, R.G., and Curti, B. J. Biol. Chem. 266, 17760–17763 (1991).

  19. 19

    Aliverti, A. et al. J. Biol. Chem. 273, 34008– 34016 (1998).

  20. 20

    Saenger, W. Principles of Nucleic Acid Structure, Springer-Verlag, New York (1983).

  21. 21

    Ammeraal, R.N., Krakow, G. & Vennesland, B. J. Biol. Chem. 240, 1820– 1823 (1965).

  22. 22

    Young, L. & Post, C.B. Biochemistry 35, 15129–15133 (1996).

  23. 23

    Batie, C.J. & Kamin, H. J. Biol. Chem. 261, 11214–11223 (1986).

  24. 24

    Strickland, S., Palmer, G. & Massey, V. J. Biol. Chem. 250, 4048– 4052 (1975).

  25. 25

    Batie, C.J. & Kamin, H. J. Biol. Chem. 259, 8832–8839 (1984).

  26. 26

    Aliverti, A. et al. Biochemistry 34, 8371– 8379 (1995).

  27. 27

    Aliverti, A. et al. Biochemistry 32, 6374– 6380 (1993).

  28. 28

    Medina, M., Martinez-Julvez, M. Hurley, J.K., Tollin, G. & Gomez-Moreno, C. Biochemistry 37 , 2715–2728 (1998).

  29. 29

    Karplus, P.A. & Bruns, C.M. J. Bioenerg. Biomembr. 26, 89–99 (1994).

  30. 30

    Krauth-Siegel, R.L., Arscott, L.D., Schönleben-Janas, A., Schirmer, R.H. & Williams, C.H., Jr. Biochemistry 37, 13968–13977 (1998).

  31. 31

    Serra, E.C., Carrillo, N., Krapp, A.R. & Ceccarelli, E.A. Protein Express. Purif. 4, 539–546 (1993).

  32. 32

    Otwinowski, Z. & Minor, W. Methods Enzymol. 276, 307–326 ( 1997).

  33. 33

    Leslie, A.G.W. CCP4 and ESF-EACMB Newsletters on Protein Crystallography 26 (1992).

  34. 34

    Collaborative Computational Project Number 4, Acta Crystallogr. D50, 760–763 (1994).

  35. 35

    Navaza, J. Acta Crystallogr. A50, 157–163 (1994).

  36. 36

    Brünger, A.T. X-PLOR, a system for crystallography and NMR, Version 3.1, Yale Univ. Press, New Haven, CT (1992).

  37. 37

    Sack, J.S. J. Molec. Graphics 6, 224–225 (1988).

  38. 38

    Kraulis, P. J. Appl. Crystallogr. 24, 946–950 (1991).

  39. 39

    McRee, D.E. J. Molec. Graphics 10, 44–46 (1992).

  40. 40

    Merrit, E.A. & Murphy, M.E.P. Acta Crystallogr. D50, 869–873 (1994).

  41. 41

    Diederichs, K. & Karplus, P.A. Nature Struct. Biol. 4, 269–275 ( 1997).

  42. 42

    Brünger, A.T. Nature 355, 472– 475 (1992).

Download references

Acknowledgements

We thank L. Piubelli for performing some of the spectroscopic experiments, S.E. Ealick for the use of his area detector facility, and T.P. Begley and V. Massey for helpful discussions. This work was supported by grants from the NSF to P.A.K., from CONICET and FONCYT (Argentina) to E.A.C., and from MURST to G.Z. N.C. was a recipient of a John Simon Guggenheim Fellowship.

Author information

Correspondence to P. Andrew Karplus.

Rights and permissions

Reprints and Permissions

About this article

Further reading