Drosophila nuclear receptor E75 is a thiolate hemoprotein.
Drosophila E75 is a member of the nuclear receptor superfamily. These eukaryotic transcription factors are involved in almost all physiological processes. They regulate transcription in response to binding of rigid hydrophobic hormone ligands. As it is the case for many nuclear receptors, the E75 hormone ligand was originally unknown. Recently, however, it was shown that the ligand binding domain (LBD) of E75 contains a tightly bound heme prosthetic group and is gas responsive. Here we have used site-directed mutagenesis along with UV-visible and electron paramagnetic resonance (EPR) spectroscopies to characterize and assign the heme iron axial ligands in E75. The F370Y mutation and addition of hemin to the growth medium during expression of the protein in Escherichia coli were necessary to produce good yields of heme-enriched E75 LBD. EPR studies revealed the presence of several species containing a strongly iron bound thiolate. The involvement of cysteines 396 and 468 in heme binding was subsequently shown by single and double mutations. Using a similar approach, we have also established that the sixth iron ligand of a well-defined coordination conformation, which accounts for approximately half of the total species, is histidine 574. The other iron coordination pairs are discussed. We conclude that E75 is a new example of a thiolate hemoprotein and that it may be involved in hormone synthesis regulation.
de Rosny
Eve
E
CEA, CNRS, UJF, UMR 5075, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France. eve.derosny@ibs.fr
de Groot
Arjan
A
Jullian-Binard
Celine
C
Gaillard
Jacques
J
Borel
Franck
F
Pebay-Peyroula
Eva
E
Fontecilla-Camps
Juan Carlos
JC
Jouve
Hélène M
HM
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D015870:Gene Expression; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006420:Hemeproteins; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; C061375:Eip75B protein, Drosophila; D006418:Heme; D006639:Histidine; D013455:Sulfur; D003545:Cysteine; D000409:Alanine