Skip to main content
SpringerLink
Log in

Relationship between the GTPase, metal-binding, and dimerization activities of E. coli HypB

  • Original Paper
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

Biosynthesis of the metallocenter in the active site of the [NiFe] hydrogenase enzyme requires the accessory protein HypB, which is a metal-binding GTPase. In this study, the interplay between the individual activities of Escherichia coli HypB was examined. The full-length protein undergoes nucleotide-responsive dimerization that is disrupted upon mutation of L242 and L246 to alanine. This mutant HypB is monomeric under all of the conditions investigated but the inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric protein has metal-binding behavior similar to that of wild-type HypB. Furthermore, expression of L242A/L246A HypB in vivo results in hydrogenase activity that is approximately half of the activity produced by the wild-type control, suggesting that dimerization of HypB does not have a critical role in the hydrogenase maturation pathway. In contrast, the GTPase activity of HypB is modulated by metal loading of the protein. These results provide insight into the role of HypB in hydrogenase biosynthesis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

AUC:

Analytical ultracentrifugation

bGDP:

Fluorescently labelled GDP analogue BODIPY FL GDP

GFC:

Gel filtration chromatography

PAR:

4-(2-Pyridylazo)resorcinol

TCEP:

Tris(2-carboxyethyl)phosphine

XAS:

X-ray absorption spectroscopy

WT:

Wild type

References

  1. Kuchar J, Hausinger RP (2004) Chem Rev 104:509–525. doi:10.1021/cr020613p

    Article  PubMed  CAS  Google Scholar 

  2. Rosenzweig AC (2002) Chem Biol 9:673–677

    Article  PubMed  CAS  Google Scholar 

  3. Mulrooney SB, Hausinger RP (2003) FEMS Microbiol Rev 27:239–261

    Article  PubMed  CAS  Google Scholar 

  4. Li Y, Zamble DB (2009) Chem Rev 109:4617–4643. doi:10.1021/cr900010n

    Article  PubMed  CAS  Google Scholar 

  5. Kaluarachchi H, Chan Chung KC, Zamble DB (2010) Nat Prod Rep 27:681–694. doi:10.1039/b906688h

    Article  PubMed  CAS  Google Scholar 

  6. Maier T, Lottspeich F, Bock A (1995) Eur J Biochem 230:133–138

    Article  PubMed  CAS  Google Scholar 

  7. Moncrief MB, Hausinger RP (1997) J Bacteriol 179:4081–4086

    PubMed  CAS  Google Scholar 

  8. Jeon WB, Cheng J, Ludden PW (2001) J Biol Chem 276:38602–38609. doi:10.1074/jbc.M104945200

    Article  PubMed  CAS  Google Scholar 

  9. Soriano A, Hausinger RP (1999) Proc Natl Acad Sci USA 96:11140–11144

    Article  PubMed  CAS  Google Scholar 

  10. Mehta N, Benoit S, Maier RJ (2003) Microb Pathog 35:229–234

    Article  PubMed  CAS  Google Scholar 

  11. Kerby RL, Ludden PW, Roberts GP (1997) J Bacteriol 179:2259–2266

    PubMed  CAS  Google Scholar 

  12. Waugh R, Boxer DH (1986) Biochimie 68:157–166

    Article  PubMed  CAS  Google Scholar 

  13. Vignais PM, Billoud B, Meyer J (2001) FEMS Microbiol Rev 25:455–501

    PubMed  CAS  Google Scholar 

  14. Leach MR, Zamble DB (2007) Curr Opin Chem Biol 11:159–165. doi:10.1016/j.cbpa.2007.01.011

    Article  PubMed  CAS  Google Scholar 

  15. Sawers G (1994) Antonie Van Leeuwenhoek 66:57–88

    Article  PubMed  CAS  Google Scholar 

  16. Menon NK, Chatelus CY, Dervartanian M, Wendt JC, Shanmugam KT, Peck HD Jr, Przybyla AE (1994) J Bacteriol 176:4416–4423

    PubMed  CAS  Google Scholar 

  17. Sauter M, Bohm R, Bock A (1992) Mol Microbiol 6:1523–1532

    Article  PubMed  CAS  Google Scholar 

  18. Bock A, King PW, Blokesch M, Posewitz MC (2006) Adv Microb Physiol 51:1–71

    Article  PubMed  Google Scholar 

  19. Winter G, Buhrke T, Lenz O, Jones AK, Forgber M, Friedrich B (2005) FEBS Lett 579:4292–4296. doi:10.1016/j.febslet.2005.06.064

    Article  PubMed  CAS  Google Scholar 

  20. Loscher S, Zebger I, Andersen LK, Hildebrandt P, Meyer-Klaucke W, Haumann M (2005) FEBS Lett 579:4287–4291. doi:10.1016/j.febslet.2005.06.063

    Article  PubMed  Google Scholar 

  21. Theodoratou E, Huber R, Bock A (2005) Biochem Soc Trans 33:108–111. doi:10.1042/BST0330108

    Article  PubMed  CAS  Google Scholar 

  22. Leach MR, Sandal S, Leach MR, Sandal S, Sun H, Zamble DB (2005) Biochemistry 44:12229–12238. doi:10.1021/bi050993j

    Article  PubMed  CAS  Google Scholar 

  23. Chan Chung KC, Cao L, Dias AV, Pickering IJ, George GN, Zamble DB (2008) J Am Chem Soc 130:14056–14057. doi:10.1021/ja8055003

    Article  Google Scholar 

  24. Gasper R, Scrima A, Wittinghofer A (2006) J Biol Chem 281:27492–27502. doi:10.1074/jbc.M600809200

    Article  PubMed  CAS  Google Scholar 

  25. Maier T, Jacobi A, Sauter M, Bock A (1993) J Bacteriol 175:630–635

    PubMed  CAS  Google Scholar 

  26. Dias AV, Mulvihill CM, Leach MR, Pickering IJ, George GN, Zamble DB (2008) Biochemistry 47:11981–11991. doi:10.1021/bi801337x

    Article  PubMed  CAS  Google Scholar 

  27. Gill SC, von Hippel PH (1989) Anal Biochem 182:319–326

    Article  PubMed  CAS  Google Scholar 

  28. Baykov AA, Evtushenko OA, Avaeva SM (1988) Anal Biochem 171:266–270

    Article  PubMed  CAS  Google Scholar 

  29. Jacobi A, Rossmann R, Bock A (1992) Arch Microbiol 158:444–451

    Article  PubMed  CAS  Google Scholar 

  30. Leach MR, Zhang JW, Zamble DB (2007) J Biol Chem 282:16177–16186. doi:10.1074/jbc.M610834200

    Article  PubMed  CAS  Google Scholar 

  31. Ballantine SP, Boxer DH (1985) J Bacteriol 163:454–459

    PubMed  CAS  Google Scholar 

  32. Zhang JW, Butland G, Greenblatt JF, Emili A, Zamble DB (2005) J Biol Chem 280:4360–4366. doi:10.1074/jbc.M411799200

    Article  PubMed  CAS  Google Scholar 

  33. Mehta N, Olson JW, Maier RJ (2003) J Bacteriol 185:726–734

    Article  PubMed  CAS  Google Scholar 

  34. Fu C, Olson JW, Maier RJ (1995) Proc Natl Acad Sci USA 92:2333–2337

    Article  PubMed  CAS  Google Scholar 

  35. Hube M, Blokesch M, Bock A (2002) J Bacteriol 184:3879–3885

    Article  PubMed  CAS  Google Scholar 

  36. Atanassova A, Zamble DB (2005) J Bacteriol 187:4689–4697. doi:10.1128/JB.187.14.4689-4697.2005

    Article  PubMed  CAS  Google Scholar 

  37. Stingl K, Schauer K, Ecobichon C, Labigne A, Lenormand P, Rousselle J-C, Namane A, De Reuse H (2008) Mol Cell Proteomics 7:2429–2441. doi:10.1074/mcp.M800160-MCP200

    Article  PubMed  CAS  Google Scholar 

  38. Leipe DD, Wolf YI, Koonin EV, Aravind L (2002) J Mol Biol 317:41–72. doi:10.1006/jmbi.2001.5378

    Article  PubMed  CAS  Google Scholar 

  39. Sydor AM, Liu J, Zamble DB (2011) J Bacteriol 193:1359–1368. doi:10.1128/JB.01333-10

    Article  PubMed  Google Scholar 

  40. Boer JL, Quiroz-Valenzuela S, Anderson KL, Hausinger RP (2010) Biochemistry 49:5859–5869. doi:10.1021/bi1004987

    Article  PubMed  CAS  Google Scholar 

  41. Jeoung JH, Giese T, Grunwald M, Dobbek H (2010) J Mol Biol 396:1165–1179. doi:10.1016/j.jmb.2009.12.062

    Article  PubMed  CAS  Google Scholar 

  42. Zambelli B, Stola M, Musiani F, De Vriendt K, Samyn B, Devreese B, Van Beeumen J, Turano P, Dikiy A, Bryant DA, Ciurli S (2005) J Biol Chem 280:4684–4695

    Article  PubMed  CAS  Google Scholar 

  43. Zambelli B, Turano P, Musiani F, Neyroz P, Ciurli S (2009) Proteins 74:222–239. doi:10.1002/prot.22205

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded in part by grants from the Canadian Institutes of Health Research and the Canada Research Chairs Program. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, M5S 3H6, Canada

    Fang Cai, Thanh T. Ngu, Harini Kaluarachchi & Deborah B. Zamble

Authors
  1. Fang Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thanh T. Ngu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Harini Kaluarachchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Deborah B. Zamble
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Deborah B. Zamble.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material (PDF 750 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cai, F., Ngu, T.T., Kaluarachchi, H. et al. Relationship between the GTPase, metal-binding, and dimerization activities of E. coli HypB. J Biol Inorg Chem 16, 857–868 (2011). https://doi.org/10.1007/s00775-011-0782-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-011-0782-y

Keywords

Search

Navigation