Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA.
Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Vanderbilt University Medical Center, Nashville, TN, USA; Vanderbilt Diabetes Research and Training Center, Vanderbilt University Medical Center, Nashville, TN, USA.
J Lipid Res. 2021;62:100081. doi: 10.1016/j.jlr.2021.100081. Epub 2021 Apr 29.
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 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.
核受体是结合脂质的转录因子,这一事件诱导受体发生结构构象变化,有利于与转录共激活因子相互作用。核受体类固醇生成因子 1(SF-1,NR5A1)结合信号转导磷脂酰肌醇 4,5-二磷酸(PI(4,5)P2,PIP2)和磷脂酰肌醇 3,4,5-三磷酸(PI(3,4,5)P3,PIP3),我们之前的晶体结构显示了磷脂酰肌醇头基如何调节 SF-1 的功能。然而,酰基链在调节 SF-1 结构中的作用仍未得到解决。在这里,我们使用体外结合和功能测定的 X 射线晶体学方法研究了 PIP3 的酰基链如何调节人 SF-1 配体结合结构域的结构和功能。改变酰基链长度和不饱和度调节所有测试的磷脂酰肌醇与 SF-1 的表观结合。基于质谱的脂质组学数据表明,C16 和 C18 磷脂优先与在细菌中异位表达的 SF-1 结合。然后,我们解决了 SF-1 与二油酰基 PIP3(18:1/18:1)结合的 2.5Å 晶体结构,将其与 SF-1 与二棕榈酰基 PIP3(16:0/16:0)结合的匹配结构进行比较。与致病的人类多态性相关的特定 SF-1 区域以及对 SF-1 功能至关重要的共激活剂结合区域内的二油酰基结合结构严重无序,同时在溶液中增加了对蛋白酶消化的敏感性。验证这些结构观察结果,体外功能研究表明,与二棕榈酰基 PIP3 相比,二油酰基 PIP3 诱导过氧化物酶体增殖物激活受体 γ 共激活因子 1-α 共激活肽与 SF-1 的结合亲和力降低 6 倍。总之,这些数据表明,磷脂酰肌醇酰基链的化学性质控制 SF-1 中特定的、临床上重要的结构区域的有序状态,调节 SF-1 的体外功能。