title: Probing conformational changes in orphan nuclear receptor: the NGFI-B intermediate is a partially unfolded dimer.
abstract: Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the alpha-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners.
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970, Brazil.
authors.forename: Wanius
authors.initials: W
authors.lastname: Garcia
authors.forename: Ana Carolina M
authors.initials: AC
authors.lastname: Figueira
authors.forename: Mario
authors.initials: M
authors.lastname: de Oliveira Neto
authors.forename: Carolina A
authors.initials: CA
authors.lastname: de Guzzi
authors.forename: Hilde H
authors.initials: HH
authors.lastname: Buzzá
authors.forename: Rodrigo V
authors.initials: RV
authors.lastname: Portugal
authors.forename: Marcos R
authors.initials: MR
authors.lastname: Calgaro
authors.forename: Igor
authors.initials: I
authors.lastname: Polikarpov
mesh_terms: D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014965:X-Rays
chemical_list: D004268:DNA-Binding Proteins; C539821:NR4A1 protein, human; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D019791:Guanidine