Waku Tsuyoshi, Shiraki Takuma, Oyama Takuji, Fujimoto Yoshito, Maebara Kanako, Kamiya Narutoshi, Jingami Hisato, Morikawa Kosuke
Takara Bio Endowed Division, Department of Biomolecular Recognition, Institute for Protein Research, Osaka University, Open Laboratories of Advanced Bioscience and Biotechnology, Suita, Osaka, Japan.
J Mol Biol. 2009 Jan 9;385(1):188-99. doi: 10.1016/j.jmb.2008.10.039. Epub 2008 Oct 19.
Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor that regulates lipid homeostasis, and several fatty acid metabolites have been identified as PPARgamma ligands. Here, we present four crystal structures of the PPARgamma ligand binding domain (LBD) covalently bound to endogenous fatty acids via a unique cysteine, which is reportedly critical for receptor activation. The structure analyses of the LBD complexed with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) revealed that the covalent binding of 15d-PGJ(2) induced conformational changes in the loop region following helix H2', and rearrangements of the side-chain network around the created covalent bond in the LBD. Point mutations of these repositioned residues on the loop and helix H3 almost completely abolished PPARgamma activation by 15d-PGJ(2), indicating that the observed structural alteration may be crucial for PPARgamma activation by the endogenous fatty acid. To address the issue of partial agonism of endogenous PPARgamma ligands, we took advantage of a series of oxidized eicosatetraenoic acids (oxoETEs) as covalently bound ligands to PPARgamma. Despite similar structural and chemical properties, these fatty acids exhibited distinct degrees of transcriptional activity. Crystallographic studies, using two of the oxoETE/PPARgamma LBD complexes, revealed that transcriptional strength of each oxoETE is associated with the difference in the loop conformation, rather than the interaction between each ligand and helix H12. These results suggest that the loop conformation may be responsible for the modulation of PPARgamma activity. Based on these results, we identified novel agonists covalently bound to PPARgamma by in silico screening and a cell-based assay. Our crystallographic study of LBD complexed with nitro-233 demonstrated that the expected covalent bond is indeed formed between this newly identified agonist and the cysteine. This study presents the structural basis for the activation and modulation mechanism of PPARgamma through covalent modification with endogenous fatty acids.
过氧化物酶体增殖物激活受体（PPAR）γ是一种调节脂质稳态的核受体，并且几种脂肪酸代谢产物已被鉴定为PPARγ配体。在此，我们展示了PPARγ配体结合域（LBD）通过一个独特的半胱氨酸与内源性脂肪酸共价结合的四种晶体结构，据报道该半胱氨酸对受体激活至关重要。与15 - 脱氧 - Δ（12,14） - 前列腺素J2（15d - PGJ2）复合的LBD的结构分析表明，15d - PGJ2的共价结合诱导了螺旋H2'之后环区域的构象变化，以及LBD中形成的共价键周围侧链网络的重排。环和螺旋H3上这些重新定位残基的点突变几乎完全消除了15d - PGJ2对PPARγ的激活，表明观察到的结构改变可能对内源性脂肪酸激活PPARγ至关重要。为了解决内源性PPARγ配体的部分激动作用问题，我们利用了一系列氧化二十碳四烯酸（oxoETEs）作为与PPARγ共价结合的配体。尽管这些脂肪酸具有相似的结构和化学性质，但它们表现出不同程度的转录活性。使用两种oxoETE/PPARγ LBD复合物进行的晶体学研究表明，每种oxoETE的转录强度与环构象的差异相关，而不是与每种配体和螺旋H12之间的相互作用相关。这些结果表明环构象可能负责PPARγ活性的调节。基于这些结果，我们通过计算机筛选和基于细胞的测定鉴定了与PPARγ共价结合的新型激动剂。我们对与硝基 - 233复合的LBD的晶体学研究表明，在这种新鉴定的激动剂和半胱氨酸之间确实形成了预期的共价键。这项研究展示了通过与内源性脂肪酸共价修饰来激活和调节PPARγ的机制的结构基础。
