Advertisement

JBIC Journal of Biological Inorganic Chemistry

, Volume 20, Issue 8, pp 1253–1261 | Cite as

Histidine residues are important for preserving the structure and heme binding to the C. elegans HRG-3 heme-trafficking protein

  • Ortal Marciano
  • Yoni Moskovitz
  • Iqbal Hamza
  • Sharon RuthsteinEmail author
Original Paper

Abstract

C. elegans is a heme auxotroph that requires environmental heme for sustenance. As such, worms utilize HRG-3, a small heme-trafficking protein, to traffic heme from the intestine to extra-intestinal tissues and embryos. However, how HRG-3 binds and delivers heme remains unknown. In this study, we utilized electron paramagnetic resonance spectroscopy together with site-directed spin labeling, absorption spectroscopy, circular dichroism, and mutagenesis to gain structural and molecular insights into HRG-3. We showed that HRG-3 is a dimer, whereas H9 and H10 are significant residues that preserve a specific conformational state in the HRG-3 dimer. In the absence of H9 and H10, HRG-3 can still bind heme, although with a different affinity. Furthermore, the heme-binding site is closer to the N-termini than to the C-termini. Taken together, our results lay the groundwork for future mechanistic and structural studies of HRG-3 and inter-tissue heme trafficking in metazoans.

Keywords

HRG-3 Heme transfer EPR Site-directed spin labeling Absorption spectroscopy 

Abbreviations

CD

Circular dichroism

CW

Continuous wave

EPR

Electron paramagnetic resonance

MTSSL

1-Oxyl-2,2,5,5-tetramethyl-2,5-pyrroline-3-methyl) methanesulfonothioate

SDSL

Site-directed spin labeling

Notes

Acknowledgments

This work was supported by funds received from Bar Ilan University.

References

  1. 1.
    Ajioka RS, Phillips JD, Kushner JP (2006) BioChem BioPhys Acta 1763:723–736CrossRefPubMedGoogle Scholar
  2. 2.
    Schultz IJ, Chen C, Paw BH, Hamza I (2010) J Biol Chem 285:26753–26759PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Severance S, Hamza I (2009) Chem Rev 109:4596–4616PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Yuan X, Fleming MD, Hamza I (2013) Curr Opin Chem Biol 17:204–211PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Yuan X, Prochenko O, Philpott CC, Hamza I (2012) J Biol Chem 287:4914–4924PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Philpott CC (2012) J Biol Chem 287:13518–13523PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Chen C, Samuel TK, Krause M, Dailey HA, Hamza I (2012) J Biol Chem 287:9601–9612PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Chen C, Samuel TK, Sinclair J, Dailey HA, Hamza I (2011) Cell 145:720–731PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Hamza I (2006) ACS Chem Biol 1:627–629PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Severance S, Rajagopal A, Rao AU, Cerqueira GC, Miterva M, El-Sayed N, Krause M, Hamza I (2010) PLoS Genet 6:e1001044PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Rao AU, Carta LK, Lesuisse E, Hamza I (2005) Proc Natl Acad Sci 102:4270–4275PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Aramini JM, Hamilton K, Rossi P, Ertekin A, Lee H-W, Lemak A, Wang H, Xiao R, Acton TB, Everett JK, Montelione GT (2012) Biochemistry 51:3705–3707PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Science 239:487–491CrossRefPubMedGoogle Scholar
  14. 14.
    Peterson GL (1997) Anal Biochem 83:346–356CrossRefGoogle Scholar
  15. 15.
    Del Vecchio M, Pogni R, Baratto MC, BNobbs A, Rappuoli R, Pizza M, Balducci E (2009) J Biol Chem 284:33040–33047Google Scholar
  16. 16.
    Peisach J, Blumberg WE, Lode ET, Coon MJ (1971) J Biol Chem 246:5877–5881PubMedGoogle Scholar
  17. 17.
    Hubbell WL, Gross A, Langen R, Lietzow MA (1998) Curr Opin Struct Biol 8:649–656CrossRefPubMedGoogle Scholar
  18. 18.
    Puljung MC, DeBerg HA, Zagotta WN, Stoll S (2014) Proc Natl Acad Sci 111:9816–9821PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Joseph B, Morkhov VM, Yulikov M, Jeschke G, Bordignon E (2014) J Biol Chem 289:3176–3185PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Atherton NM (1993) Principles of electron spin resonance. Ellis Horwood PTR Prentice Hall, EnglandGoogle Scholar
  21. 21.
    Weil JA, Bolton JR (2007) Electron paramagnetic resonance. Wiley, HobokenGoogle Scholar
  22. 22.
    Eaton G, Eaton SS (1989) In biological magnetic resonance. Plenum, New YorkGoogle Scholar
  23. 23.
    Freed JH (1976) In spin labeling (theory and applications). Academic Press, New YorkGoogle Scholar
  24. 24.
    Gorcester J, Millhauser GL, Freed JH (1990) In modern pulsed and continuous wave electron spin resonance. Wiley, New YorkGoogle Scholar
  25. 25.
    McConnell HM (1998) In: Foundations of modern EPR. World Scientific, SingaporeGoogle Scholar
  26. 26.
    Budil DE, Lee S, Saxena S, Freed JH (1996) J Magn Reson Ser A 120:155–189CrossRefGoogle Scholar
  27. 27.
    Klare JP (2013) Biol Chem 394:1281–1300CrossRefPubMedGoogle Scholar
  28. 28.
    Huynh C, Yuan X, Miguel DC, Renberg RL, Protchenko O, Philpott CC, Hamza I, Andrews NW (2012) PLoS Pathog 8:e1002795PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Yoo B-K, Lamarre I, Martin J-L, Rappaport F, Negrerie M (2015) Proc Natl Acad Sci 112:E1697–E1704PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Yoo B-K, Lamarre I, Martin J-L, Andrew CR, Negrerie M (2013) J Am Chem Soc 135:3248–3254CrossRefPubMedGoogle Scholar
  31. 31.
    Vidossich P, Magistrato A (2014) Biomolecules 4:616–645PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Benitez JJ, Keller AM, Huffman DL, Yatsunyk LA, Rosenzweig AC, Chen P (2011) Faraday Discuss 148:71–82PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Bohm G, Muhr R, Jaenicke R (1992) Protein Eng 5:191–195CrossRefPubMedGoogle Scholar
  34. 34.
    Berlow RB, Dyson HJ, Wright PE (2015) FEBS Lett. 589:2433–2440CrossRefPubMedGoogle Scholar

Copyright information

© SBIC 2015

Authors and Affiliations

  • Ortal Marciano
    • 1
  • Yoni Moskovitz
    • 1
  • Iqbal Hamza
    • 2
    • 3
  • Sharon Ruthstein
    • 1
    Email author
  1. 1.Department of Chemistry, Faculty of Exact SciencesBar Ilan UniversityRamat-GanIsrael
  2. 2.Department of Animal and Avian SciencesUniversity of MarylandCollege ParkUSA
  3. 3.Department of Cell Biology and Molecular GeneticsUniversity of MarylandCollege ParkUSA

Personalised recommendations