title: Histidine residues are important for preserving the structure and heme binding to the C. elegans HRG-3 heme-trafficking protein.
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.
authors.affiliation: Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel.
authors.forename: Ortal
authors.initials: O
authors.lastname: Marciano
authors.affiliation: Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel.
authors.forename: Yoni
authors.initials: Y
authors.lastname: Moskovitz
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park, MD, 20742, USA.|Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA.
authors.forename: Iqbal
authors.initials: I
authors.lastname: Hamza
authors.affiliation: Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel. sharon.ruthstein@biu.ac.il.
authors.forename: Sharon
authors.initials: S
authors.lastname: Ruthstein
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D029742:Caenorhabditis elegans Proteins; D006420:Hemeproteins; D006639:Histidine
keywords: Absorption spectroscopy; EPR; HRG-3; Heme transfer; Site-directed spin labeling