Solution Nuclear Magnetic Resonance Studies of the Ligand-Binding Domain of an Orphan Nuclear Receptor Reveal a Dynamic Helix in the Ligand-Binding Pocket

Abstract

The ligand-binding domains (LBDs) of the NR5A subfamily of nuclear receptors activate transcription via ligand-dependent and ligand-independent mechanisms. The Drosophila Ftz-F1 receptor (NR5A3) belongs to the latter category, and its ligand independence is attributed to a short helical segment (α6) within the protein that resides in the canonical ligand-binding pocket (LBP) in the crystalline state. Here, we show that the α6 helix is dynamic in solution when Ftz-F1 is bound to the LxxLL motif of its cofactor Ftz, undergoing motions on the fast (picosecond to nanosecond) as well as slow (microsecond to millisecond) time scales. Motions on the slow time scale (∼10 s) appear to pervade throughout the domain, most prominently in the LBP and residues at or near the cofactor-binding site. We ascribe the fast time scale motions to a solvent-accessible conformation for the α6 helix akin to those described for its orthologs in higher organisms. We assign this conformation where the LBP is "open" to a lowly populated species, while the major conformer bears the properties of the crystal structure where the LBP is "closed". We propose that these conformational transitions could allow binding to small molecule ligands and/or play a role in dissociation of the cofactor from the binding site. Indeed, we show that the Ftz-F1 LBD can bind phospholipids, not unlike its orthologs. Our studies provide the first detailed insights into intrinsic motions occurring on a variety of time scales in a nuclear receptor LBD and reveal that potentially functionally significant motions pervade throughout the domain in solution, despite evidence to the contrary implied by the crystal structure.

The Intrinsically Disordered Region of GCE Protein Adopts a More Fixed Structure by Interacting With the LBD of the Nuclear Receptor FTZ-F1

“…We decided to analyze possible interactions of this domain with short peptide GCEC PEP (LRLIQNLQK), corresponding to the LIXXL motif present in the GCEC sequence [22]. The recorded reference spectrum is similar to the spectrum presented by Daffern et al [23], and shows a good peaks dispersion appropriate for globular protein (Fig. 8, blue).…”

“…As demonstrated, GCE and MET are able to interact with the transcription factor Fushi Tarazu factor-1 (FTZ-F1) [22]. Drosophila FTZ-F1 plays a critical role in the development of the segmented body plan in the embryo [23] and allows the crosstalk of the 20E and JH signaling pathways. In the absence of JH, FTZ-F1 binds to the 20E receptor composed of two proteins: the Ultraspiracle (Usp) and Ecdysone receptor (Ecr), and contributes to the induction of metamorphosis.…”

The intrinsically disordered region of GCE protein adopts a more fixed structure by interacting with the LBD of the nuclear receptor FTZ-F1

“…We decided to analyze possible interactions of this domain with short peptide GCEC PEP (LRLIQNLQK), corresponding to the LIXXL motif present in the GCEC sequence [18]. The recorded reference spectrum is similar to the spectrum presented by Daffern et al [19], and shows a good peaks dispersion appropriate for globular protein (Fig. 8, blue).…”

“…As demonstrated, GCE and MET are able to interact with the transcription factor Fushi Tarazu factor-1 (FTZ-F1) [18]. Drosophila FTZ-F1 plays a critical role in the development of the segmented body plan in the embryo [19] and allows the crosstalk of the 20E and JH signaling pathways. In the absence of JH, FTZ-F1 binds to the 20E receptor composed of two proteins: the Ultraspiracle (Usp) and Ecdysone receptor (Ecr), and contributes to the induction of metamorphosis.…”

Orphan nuclear receptorftz-f1 (NR5A3)promotes egg chamber survival in theDrosophilaovary

“…The mammalian NR5A subgroup of nuclear receptors has been implicated in a variety of reproductive contexts, including sex determination, gonad development, and ovulation ( Yazawa et al 2015 ; Meinsohn et al 2019 ). NR5A receptors are considered “orphan” nuclear receptors, able to bind phospholipids but also adopt an active conformation in the absence of a ligand ( Krylova et al 2005 ; Yoo et al 2011 ; Lu et al 2013 ; Musille et al 2013 ; Daffern et al 2018 ). Mammalian NR5A members Liver Receptor Homolog 1 (LRH-1) and Steroidogenic Factor-1 (SF-1) bind the same DNA sequence motif but regulate distinct sets of target genes in multiple tissues associated with the reproductive axis ( Meinsohn et al 2019 ).…”