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Crystal structure of the anti-fungal target N-myristoyl transferase

Nature Structural Biology volume 5, pages 213–221 (1998)Cite this article

Abstract

N-myristoyl transferase (NMT) catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins, and is found only in eukaryotic cells. The enzyme in this study is the 451 amino acid protein produced by Candida albicans, a yeast responsible for the majority of systemic infections in immuno-compromised humans. NMT activity is essential for vegetative growth, and the structure was determined in order to assist in the discovery of a selective inhibitor of NMT which could be developed as an anti-fungal drug. NMT has no sequence homology with other protein sequences and has a novel α/β fold which shows internal twofold symmetry, which may be a result of gene duplication. On one face of the protein there is a long, curved, relatively uncharged groove, at the center of which is a deep pocket. The pocket floor is negatively charged due to the vicinity of the C-terminal carboxylate and a nearby conserved glutamic acid residue, which separates the pocket from a cavity. These observations, considered alongside the positions of residues whose mutation affects substrate binding and activity, suggest that the groove and pocket are the sites of substrate binding and the floor of the pocket is the catalytic center.

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References

  1. Bhatnagar, R.S. & Gordon, J.I. Understanding covalent modifications of proteins by lipids: where cell biology and biophysics mingle. Trends Cell Biol. 7, 14–20 (1997)

    Article  CAS  Google Scholar 

  2. Boutin, J.A. Myristoylation Cell Signal 9, 15–35 (1997).

    Article  CAS  Google Scholar 

  3. Rudnick, D.A., McWherter, C.A., Gokel, G.W. & Gordon, J.I., Myristoyl-CoA: Protein N-myristoyl transferase. Adv. Enzymol. 67, 375–430 (1993).

    CAS  PubMed  Google Scholar 

  4. Johnson, D.R., Bhatnagar, R.S., Knoll, L.J. & Gordon, J.I. Genetic and biochemical studies of protein N-myristoylation. Ann. Rev. Biochem. 63, 869–914 (1994).

    Article  CAS  Google Scholar 

  5. Boutin, J.A. et al. Myristoyl-CoA : protein N-myristoyl transferase activity in cancer cells. Purification and characterisation of a cytosolic isoform from the murine leukaemia cell line L1210. Eur. J. Biochem 214, 853–867 (1993).

    Article  CAS  Google Scholar 

  6. Resh, M.D. Interaction of tyrosine kinase oncoproteins with cellular membranes. Biochem. Biophys. Acta 1115, 307–322 (1993).

    Google Scholar 

  7. Knoll, L.J., Russell Johnson, D., Bryant, M.L. & Gordon, J.I. “Functional Significance of Myristoyl Moiety in N-Myristoyl Proteins” in Lipid Modifications of Proteins eds. Casey, P.J. & Buss, J.E. Methods in Enzymology 250, 405–435 (1995).

    Article  CAS  Google Scholar 

  8. Rudnick, D.A. et al. Kinetic and structural evidence for a sequential ordered Bi Bi mechanism of catalysis by Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyl transferase. J. Biol. Chem. 266, 9732–9739 (1991).

    CAS  PubMed  Google Scholar 

  9. Rudnick, D.A., Johnson, R.L. & Gordon, J.I. Studies of the catalytic activities and substrate specificities of Saccharomyces cerevisiae myristoyl-coenzyme A: protein N-myristoyl transferase deletion mutants and human/yeast NMT chimeras in Escherichia coli and Saccharomyces cerevisiae. J. Biol. Chem. 267, 23852–23861 (1992).

    CAS  PubMed  Google Scholar 

  10. Zhang, L., Jackson-Machelski, E. & Gordon, J.I. Biochemical studies of Saccharomyces cerevisiae myristoyl-coenzyme A : protein N-myristoyl transferase mutants. J. Biol. Chem. 271, 33131–33140 (1996).

    Article  CAS  Google Scholar 

  11. Peseckis, S.M. & Resh, M.D. Fatty acyl transfer by human N-myristoyl transferase is dependent upon conserved cysteine and histidine residues J. Biol. Chem. 269, 30888–30892 (1994).

    CAS  PubMed  Google Scholar 

  12. Duronio, R.J., Towler, D.A., Heuckeroth, R.O. & Gordon, J.I. Disruption of the yeast N-myristoyl transferase gene causes recessive lethality. Science 243, 796–800 (1989).

    Article  CAS  Google Scholar 

  13. Duronio, R.J., Reed, S.I. & Gordon, J.I. Mutations of human myristoyl-CoA : protein N-myristoyl transferase cause temperature-sensitive myristic acid auxotrophy in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 89, 4129–4133 (1992).

    Article  CAS  Google Scholar 

  14. Wiegand, R.C. et al. The Candida albicans myristoyl-CoA : protein N-myristoyl transferase gene. Isolation and expression in Saccharomyces cerevisiae and Escherichia coli. J. Biol. Chem. 267, 8591–8598 (1992).

    CAS  PubMed  Google Scholar 

  15. Lodge, J.K., Johnson, R.L., Weinberg, R.A. & Gordon, J.I. Comparison of myristoyl-CoA : protein N-myristoyl transferases from three pathogenic fungi: Cryptococcus neoformans, Histoplasma capsulatum, and Candida albicans. J. Biol. Chem. 269, 2996–3009 (1994).

    CAS  PubMed  Google Scholar 

  16. Ntwasa, M., Egerton, M. & Gay, N.J. Sequence and expression of Drosophila myristoyl-CoA : protein N-myristoyl transferase: evidence for proteolytic processing and membrane localisation. J Cell. Sci. 110, 149–156 (1997).

    CAS  PubMed  Google Scholar 

  17. Rudnick, D.A., Lu, T., Jackson-Machelski, E., Hernandez, J.C., Li, Q., Gokel, G.W. & Gordon, J.I. Analogs of palmitoyl-CoA that are substrates for myristoyl-CoA : protein N-myristoyl transferase. Proc. Natl. Acad. Sci. USA 89, 10507–10511 (1992).

    Article  CAS  Google Scholar 

  18. Duronio, R.J., Rudnick, D.A., Adams, S.P., Towler, D.A. & Gordon, J.I. Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyl transferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli. J. Biol. Chem. 266, 10498–10504 (1991).

    CAS  PubMed  Google Scholar 

  19. McWherter, C.A. et al. Scanning alanine mutagenesis and de-peptidization of a Candida albicans myristoyl-CoA : protein N-myristoyl transferase octapeptide substrate reveals three elements critical for molecular recognition. J. Biol. Chem. 272, 11874–11880 (1997).

    Article  CAS  Google Scholar 

  20. Langner, C.A. et al. 4-oxatetradecanoic acid is fungicidal for Cryptococcus neoformans and inhibits replication of human immunodeficiency virus I. J. Biol. Chem. 267, 17159–17169 (1992).

    CAS  PubMed  Google Scholar 

  21. Weinberg, R.A., McWherter, C.A., Freeman, S.K., Wood, D.C., Gordon, J.I. & Lee, S.C. Genetic studies reveal that myristoyl-CoA : protein N-myristoyl transferase is an essential enzyme in Candida albicans. Mol. Microbiol. 16, 241–250 (1995).

    Article  CAS  Google Scholar 

  22. Nagaragan, S.R. et al. Conformationally constrained [p-(omega-aminoalkyl)phenacetyl]-L-seryl-L-lysyl dipeptide amides as potent peptidomimetic inhibitors of Candida albicans and human myristoyl-CoA : protein N-myristoyl transferase. J. Med. Chem. 40, 1422–1438 (1997).

    Article  Google Scholar 

  23. de La Fortelle, E. & Bricogne, G. Maximum-Likelihood Heavy-Atom Parameter Refinement in the MIR and MAD Methods. Meth. Enz. 276, 472–494 (1997).

    Article  CAS  Google Scholar 

  24. Holm, L. & Sander, C. New Structure - Novel Fold? Structure 5, 165–171 (1997).

    Article  CAS  Google Scholar 

  25. Kleywegt, G.J. & Jones, T.A. Detecting folding motifs and similarities in protein structures. Meth. Enz. 277, 525–545 (1997).

    Article  CAS  Google Scholar 

  26. Murray-Rust, J. et al. Topological similarities in TGFβ2, PDGF-BB and NGF define a superfamily of polypeptide growth factors. Structure 1, 153–159 (1993).

    Article  CAS  Google Scholar 

  27. Cameron, O.D., Olin, B., Ridderstrom, M., Mannervik, B. & Jones, T.A. Crystal structure of human glyoxalase. 1. Evidence for gene duplication and domain swapping. EMBO J. 16, 3386–3395 (1997).

    Article  CAS  Google Scholar 

  28. Conti, E., Franks, N.P. & Brick, P. Crystal structure of luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4, 287–298 (1996).

    Article  CAS  Google Scholar 

  29. Engel, C. & Wierenga, R. The diverse world of coenzyme A binding proteins. Curr. Opin. Struct. Biol. 6, 790–797 (1996).

    Article  CAS  Google Scholar 

  30. Nicholls, A., Sharp, K. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).

    Article  CAS  Google Scholar 

  31. Kleywegt, G.J. & Jones, T.A. Detection, Delineation, Measurement and Display of Cavities in Macromolecular Structures. Acta Crystallogr. D50, 178–185 (1994).

    CAS  Google Scholar 

  32. Bairoch, A., Bucher, P. & Hofmann, K. The PROSITE database, its status in 1997. Nucleic Acids Res. 25, 217–221 (1997).

    Article  CAS  Google Scholar 

  33. Wierenga, R.K., Drenth, J. & Schultz, G.E. Comparison of the three-dimensional protein and nucleotide structure of the FAD-binding domain of p-hydroxybenzoate hydroxylase with the FAD- as well as NADPH-binding domains of glutathione reductase. J. Mol. Biol. 167, 725–739 (1983).

    Article  CAS  Google Scholar 

  34. Rudnick, D.A. et al. Structural and functional studies of Saccharomyces cerevisiae myristoyl-CoA : protein N-myristoyl transferase produced in Escherichia coli. Evidence for an acyl-enzyme intermediate. J. Biol. Chem. 265, 13370–13378 (1990).

    CAS  PubMed  Google Scholar 

  35. Quilin, M., Baase, W. & Matthews, B. Collected Abstracts of the XVII Congress and General Assembly of the International Union of Crystallography, Seattle (Wa), USA. (ed. J.F. Griffin) C-215 Acta Crystallogr. A52 (1996).

    Google Scholar 

  36. Schiltz, M., Fourme, R., Broutin, I. & Prange, T. The catalytic site of serine proteinases as a specific binding cavity for xenon. Structure 3, 309–316 (1995).

    Article  CAS  Google Scholar 

  37. Schiltz, M., Prangé, T. & Fourme, R. On the preparation and X-ray data collection of isomorphous xenon derivatives. J. Appl. Cryst. 27, 950–960 (1994).

    Article  CAS  Google Scholar 

  38. Rocque, W.J., McWherter, C.A., Wood, D.C. & Gordon, J.I. A comparative analysis of the kinetic mechanism and peptide substrate specificity of human and Saccharomyces cerevisiae myristoyl-CoA : protein N-myristoyl transferase.J. Biol. Chem. 268, 9964–9971 (1993).

    CAS  PubMed  Google Scholar 

  39. Mcllhinney, R.A., Patel, P.B. & McGlone, K. Characterization of a polyhistidine-tagged form of human myristoyl-CoA: protein N-myristoyl transferase produced in Escherichia coli. Eur. J. Biochem. 222, 137–146 (1994).

    Article  Google Scholar 

  40. Bhatnagar, R.S., Jackson-Machelski, E., McWherter, C.A. & Gordon, J.I. Isothermal titration calorimetric studies of Saccharomyces cerevisiae myristoyl-CoA : protein N-myristoyl transferase. Determinants of binding energy and catalytic discrimination among acyl-CoA and peptide ligands. J. Biol. Chem. 269, 11045–11053 (1994).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  42. Leslie, A.G.W. Molecular Data Processing Crystallographic Computing 5 (Moras, D., Podjarny, A. D. & Thierry, J. C., eds.) (Oxford University Press; 1990).

    Google Scholar 

  43. Collaborative Computing Project No. 4: The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D50, 760–763 (1994).

  44. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  45. Otwinowski, Z., In Proceedings of the CCP4 Study Weekend: Data Collection and Processing, 29–30 January 1993. (Compiled by: L. Sawyer, N. Isaacs and S. Bailey) 56–62 (SERC Daresbury Laboratory, England; 1993).

  46. Stowell, M.H.B. et al. A simple device for studying macromolecular crystals under moderate gas pressures (0.1–10 MPa). J. Appl. Crystallogr. 29, 608–613 (1996).

    Article  CAS  Google Scholar 

  47. Sheldrick, G.M. Phase annealing in SHELX-90: direct methods for larger structures. Acta Crystallogr. A46, 467–473 (1990).

    Article  CAS  Google Scholar 

  48. Bricogne, G. A Bayesian statistical theory of the phase problem. I. A multichannel maximum-entropy formalism for constructing generalised joint probability distributions of structure factors. Acta Crystallogr. A44, 517–545 (1988).

    Article  CAS  Google Scholar 

  49. Kleywegt, G.J. & Jones, T.A. Use of non-crystallographic symmetry in protein structure refinement. Acta Crystallogr. D52, 842–857 (1996).

    CAS  Google Scholar 

  50. Jones, T.A., Zou, J.-Y., Cowan, S.W. and Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47, 110–119 (1991).

    Article  CAS  Google Scholar 

  51. Brünger, A.T., Kuriyan, J., Karplus M. Crystallographic R-factor refinement by molecular dynamics. Science 235, 458–460 (1988).

    Article  Google Scholar 

  52. Brünger, A.T. Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475 (1992).

    Article  Google Scholar 

  53. 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 

  54. 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 

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Author notes

  1. Alec D. Tucker

    Present address: Discovery Chemistry Department, Pfizer Central Research, Sandwich, Kent, CT13 9NJ, UK

Authors and Affiliations

  1. Zeneca Pharmaceuticals, Alderley Park, Macclesfield, SK 10 4TG, UK

    Simon A. Weston, Roger Camble, Jeremy Colls, Gina Rosenbrock, Ian Taylor, Mark Egerton, Alec D. Tucker, Alan Tunnicliffe, Anil Mistry & Richard A. Pauptit

  2. Laboratory of Molecular Biology of the Medical Research Council, MRC Centre, Hills Road, Cambridge, CB2 2QH, UK

    Filippo Mancia, Eric de la Fortelle, John Irwin & Gerard Bricogne

  3. Inst. für Molekular Biotechnologie, Beutenbergstrasse 11, D-07708, Jena, Germany

    Alan Tunnicliffe

  4. Department of Biochemistry & Molecular Biophysics, Columbia University, 2960 Broadway, New York, 10027-6902, USA

    Filippo Mancia

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Weston, S., Camble, R., Colls, J. et al. Crystal structure of the anti-fungal target N-myristoyl transferase. Nat Struct Mol Biol 5, 213–221 (1998). https://doi.org/10.1038/nsb0398-213

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