Mosure Sarah A, Strutzenberg Timothy S, Shang Jinsai, Munoz-Tello Paola, Solt Laura A, Griffin Patrick R, Kojetin Douglas J
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, FL 33458, USA.
Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, FL 33458, USA.
Sci Adv. 2021 Jan 27;7(5). doi: 10.1126/sciadv.abc6479. Print 2021 Jan.
Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBβ ligand-binding domain (LBD). We solved two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.
血红素是组成型抑制性视黄酸受体相关孤儿受体（REV-ERB）核受体REV-ERBα（NR1D1）和REV-ERBβ（NR1D2）的内源性配体，但血红素如何调节REV-ERB活性仍不清楚。细胞研究表明，血红素是REV-ERB结合共抑制因子NCoR并抑制转录所必需的。然而，基于荧光的生化分析表明血红素会取代NCoR；在此，我们表明这是由于血红素依赖性假象。使用等温滴定量热法（ITC）和核磁共振光谱法，我们表明血红素结合重塑了NCoR受体相互作用结构域（RID）与REV-ERBβ配体结合结构域（LBD）结合的热力学相互作用图谱。我们解析了REV-ERBβ LBD与血红素和NCoR肽共结合的两个晶体结构，揭示了血红素依赖性NCoR结合模式。ITC和化学交联质谱分析揭示了2:1的LBD:RID化学计量比，这与细胞研究结果一致，即显示REV-ERB转录的NCoR依赖性抑制发生在二聚体DNA反应元件上。我们的研究结果应有助于在理解血红素依赖性REV-ERB活性方面取得新的进展。
