Abstract
AbstractNuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators. The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) binds the signaling phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), and our previous crystal structures showed how the phosphoinositide headgroups regulate SF-1 function. However, what role the acyl chains play in regulating SF-1 structure remains unaddressed. Here, we used X-ray crystallography with in vitro binding and functional assays to examine how the acyl chains of PIP3 regulate human SF-1 ligand-binding domain structure and function. Altering acyl chain length and unsaturation regulates apparent binding of all tested phosphoinositides to SF-1. Mass spectrometry–based lipidomics data suggest C16 and C18 phospholipids preferentially associate with SF-1 expressed ectopically in bacteria. We then solved the 2.5 Å crystal structure of SF-1 bound to dioleoyl PIP3(18:1/18:1) to compare it with a matched structure of SF-1 bound to dipalmitoyl PIP3(16:0/16:0). The dioleoyl-bound structure was severely disordered in a specific SF-1 region associated with pathogenic human polymorphisms and within the coactivator-binding region critical for SF-1 function while inducing increased sensitivity to protease digestion in solution. Validating these structural observations, in vitro functional studies showed dioleoyl PIP3 induced 6-fold poorer affinity of a peroxisome proliferator-activated receptor gamma coactivator 1-alpha coactivator peptide for SF-1 compared with dipalmitoyl PIP3. Together, these data suggest the chemical nature of the phosphoinositide acyl chains controls the ordered state of specific, clinically important structural regions in SF-1, regulating SF-1 function in vitro.
Highlights
Nuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators
We first asked if apparent binding of phosphoinositides to SF-1 ligand binding domain (LBD) is affected by acyl chain length when the headgroup is held constant, using the same mobility shift assay we used in previous studies [21, 25, 29, 38]
When holding the PI[4,5]P2 (PIP2) headgroup species constant while varying acyl chain length and unsaturation, the same pattern holds: The apparent Kd for PIP2 binding to SF-1 directly correlated in rank order with increasing PIP2 acyl chain length and degree of unsaturation: stearoyl/ arachidonoyl (18:0/20:4) 65 ± 8 nM < dioleoyl (18:1/18:1) 179 ± 24 nM < dipalmitoyl (16:0/16:0) 348 ± 42 nM
Summary
Nuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators. The dioleoyl-bound structure was severely disordered in a specific SF-1 region associated with pathogenic human polymorphisms and within the coactivator-binding region critical for SF-1 function while inducing increased sensitivity to protease digestion in solution Validating these structural observations, in vitro functional studies showed dioleoyl PIP3 induced 6-fold poorer affinity of a peroxisome proliferator-activated receptor gamma coactivator 1-alpha coactivator peptide for SF-1 compared with dipalmitoyl PIP3. Together, these data suggest the chemical nature of the phosphoinositide acyl chains controls the ordered state of specific, clinically important structural regions in SF-1, regulating SF-1 function in vitro. Wakelam, whose endless curiosity on how SF-1 might be regulated by acyl chain character wholly inspired the work
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