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

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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.

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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

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Acknowledgments

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

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Authors and Affiliations

  1. Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel

    Ortal Marciano, Yoni Moskovitz & Sharon Ruthstein

  2. Department of Animal and Avian Sciences, University of Maryland, College Park, MD, 20742, USA

    Iqbal Hamza

  3. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA

    Iqbal Hamza

Authors
  1. Ortal Marciano
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  2. Yoni Moskovitz
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  3. Iqbal Hamza
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  4. Sharon Ruthstein
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Correspondence to Sharon Ruthstein.

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Marciano, O., Moskovitz, Y., Hamza, I. et al. Histidine residues are important for preserving the structure and heme binding to the C. elegans HRG-3 heme-trafficking protein. J Biol Inorg Chem 20, 1253–1261 (2015). https://doi.org/10.1007/s00775-015-1304-0

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  • DOI: https://doi.org/10.1007/s00775-015-1304-0

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