title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor.
abstract: We have recombinantly expressed and purified the ligand binding domains (LBDs) of four insect nuclear receptors of the E75 family. The Drosophila melanogaster and Bombyx mori nuclear receptors were purified as ferric hemoproteins with Soret maxima at 424 nm, whereas their ferrous forms had a Soret maximum at 425 nm that responds to (•)NO and CO binding. In contrast, the purified LBD of Oncopeltus fasciatus displayed a Soret maximum at 415 nm for the ferric protein that shifted to 425 nm in its ferrous state. Binding of (•)NO to the heme moiety of the D. melanogaster and B. mori E75 LBD resulted in the appearance of a peak at 385 nm, whereas this peak appeared at 416 nm in the case of the O. fasciatus hemoprotein, resembling the behavior displayed by its human homologue, Rev-erbβ. High-performance liquid chromatography analysis revealed that, unlike the D. melanogaster and B. mori counterparts, the heme group of O. fasciatus is covalently attached to the protein through the side chains of two amino acids. The high degree of sequence homology with O. fasciatus E75 led us to clone and express the LBD of Blattella germanica, which established that its spectral properties closely resemble those of O. fasciatus and that it also has the heme group covalently bound to the protein. Hence, (•)NO/CO regulation of the transcriptional activity of these nuclear receptors might be differently controlled among various insect species. In addition, covalent heme binding provides strong evidence that at least some of these nuclear receptors function as diatomic gas sensors rather than heme sensors. Finally, our findings expand the classes of hemoproteins in which the heme group is normally covalently attached to the polypeptide chain.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: Clara
authors.initials: C
authors.lastname: Aicart-Ramos
authors.forename: Margarita
authors.initials: M
authors.lastname: Valhondo Falcón
authors.forename: Paul R
authors.initials: PR
authors.lastname: Ortiz de Montellano
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rodriguez-Crespo
mesh_terms: D000818:Animals; D012831:Bombyx; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C087173:E75 protein, insect; D019476:Insect Proteins; D011987:Receptors, Steroid; D009569:Nitric Oxide; D006418:Heme