Structure of REV-ERBβ Ligand-binding Domain Bound to a Porphyrin Antagonist

Abstract

Background: REV-ERB activity is regulated by the endogenous porphyrin agonist, heme. Results: REV-ERB binds other porphyrin ligands, including CoPP and ZnPP, which function as REV-ERB antagonists. Conclusion: Differential regulation of REV-ERBs by porphyrins with different metal centers indicates that REV-ERB function is sensitive to the metal center. Significance: Antagonist porphyrin ligands may be useful chemical tools for probing the function of REV-ERBs.

Defining a canonical ligand-binding pocket in the orphan nuclear receptor Nurr1

“…Furthermore, our 19 F NMR studies show that the pocket populates two or three low energy conformations, one of which could correspond to the crystallized conformation. As mentioned above, a similar situation was observed in structural studies of the REV-ERBβ LBD, where NMR revealed the apo-REV-ERBβ pocket is dynamic on the μs-ms timescale (Matta-Camacho et al, 2014), and crystal structures revealed a dramatic expansion of the collapsed REV-ERB ligand-binding pocket to enable binding of heme (Pardee et al, 2009;(Woo et al, 2007). Crystallography studies on estrogenrelated receptor (ERR) also suggest considerable pocket adaptability, as the pocket volumes in ERR crystal structures span 40-1020 Å 3 (Gallastegui et al, 2015).…”

“…Although a crystal structure of ligand-free (apo)-REV-ERBβ revealed a collapsed pocket (Woo et al, 2007), a subsequent crystal structure revealed its putative pocket dra-matically expands by 600 Å 3 to accommodate binding of the porphyrin heme (Gallastegui et al, 2015;(Pardee et al, 2009), which was identified as an endogenous REV-ERB ligand (Raghuram et al, 2007;(Yin et al, 2007). Using NMR spectroscopy we showed that the ligand-free REV-ERBβ ligand-binding pocket is dynamic (Matta-Camacho et al, 2014), confirming the crystallography observations that its apo-pocket has the ability to expand. Furthermore, NMR studies have revealed dynamic apo-pockets for other non-orphan nuclear receptors with large crystallized pockets that bind endogenous ligands, including PPARγ (Hughes et al, 2012;(Johnson et al, 2000), RXRα (Lu et al, 2006), and VDR (Singarapu et al, 2011).…”

Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

“…Titration of NCoR ID peptide into 15 N-labeled REV-ERBβ LBD can 1) validate that the interaction occurs in the presence of heme, 2) qualitatively reflect the strength of the interaction relative to apo (ligand-free)-REV-ERBβ LBD, and 3) highlight the residues most likely involved in binding (16). We previously obtained backbone NMR chemical shift assignments for REV-ERBβ LBD (17).…”

“…The human REV-ERBβ ligand binding domain (LBD; residues 381-579) was previously cloned into the pET46 vector (17). The mouse NCoR Receptor Interaction Domain (RID; residues 1942-2208 in the X50 splice variant), which has 87% sequence identity to human NCoR RID (100% sequence identity in the ID motifs), was previously cloned into the pET32 vector (23).…”

Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes

“…CYP17A1 with the inhibitor abiraterone was used for all subsequent studies because this was the most challenging protein to produce with exogenous FePPIX supplementation and because solution NMR was a comprehensive viable method for readily determining any changes in the overall structure that might result from metal substitution. Cobalt-containing PPIX (CoPPIX) was the first metal substitution employed because it demonstrated retention of the overall structure upon incorporation of CoPPIX into myoglobin (18), the ability to coordinate to a Cys side chain as observed for FePPIX in native P450 enzymes (19), and similarity to iron in being able to bind H 2 O (20), O 2 (21), and nitrogen heterocycles (20) like that of the CYP17A1 abiraterone inhibitor.…”