Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation.
Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation.
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DOI：
10.1371/journal.pcbi.1004981
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发表时间：
2016-06
影响因子：
4.3
通讯作者：
Bonati L
中科院分区：
文献类型：
--
作者：
Corrada D;Soshilov AA;Denison MS;Bonati L
芳烃受体（AhR）是一种转录因子，介导对外源性物质的生化反应和许多环境污染物（包括二恶英）的毒性作用。最近，内源性调节作用的AhR在正常的生理和发展也有报道，从而扩大了兴趣，了解其激活的分子机制。由于AhR需要通过螺旋-环-阻碍（HLH）和PER-ARNT-SIM（PAS）结构域与AhR核转运蛋白（ARNT）发生二聚化以将AhR转化为其转录活性形式，因此破译AhR：ARNT二聚化模式将提供对AhR转化机制的见解。在这里，我们提出了同源模型的小鼠AhR：ARNT PAS结构域二聚体开发使用最近可用的X-射线结构的其他bHLH-PAS蛋白二聚体。由于不同模板二聚体中的相互取向和相互作用表面不同，针对PAS-A和PAS-B二聚体开发了两种替代模型，并且通过结合许多计算评估来表征它们。两个完善的热点预测方法和新的方法来分析单个残基和残基成对的MM-GBSA结合自由能的贡献，通过预测残基的二聚体稳定的关键。在此基础上，设计了一种针对小鼠AhR和ARNT蛋白的诱变策略，并评估了AhR：ARNT异二聚体突变体的配体依赖性DNA结合能力。虽然功能分析不支持基于HIF 2 α：ARNT异源二聚体的PAS-B模型，但PAS-A和PAS-B的基于CLOCK：BMAL 1的AhR：ARNT二聚体模型衍生的大多数突变体显著降低了DNA结合水平，表明后一种模型最适合描述AhR：ARNT二聚化。这些新的结果开辟了新的研究方向，重点是阐明AhR功能活性的基本分子机制。计算建模与实验验证相结合，可以深入了解蛋白质系统的结构和功能特性。基本的螺旋环-阻碍因子-ARNT-SIM（bHLH-PAS）蛋白显示保守的功能结构域，尽管由不同的系统发挥广泛的功能。在这个蛋白质家族中，已知芳烃受体（AhR）介导许多环境污染物的毒性作用，包括二恶英和二恶英样化学物质，并且它还发挥其他生物化学和生理学作用。尽管没有实验确定的结构，理论模型的AhR PAS结构域的基础上开发的同源系统允许了解其功能的分子机制的某些方面。在这项工作中，我们提出了替代结构模型的转录活性复合物的AhR与AhR核转位蛋白（ARNT）。建模的蛋白质-蛋白质相互作用界面的计算表征指导诱变实验的设计，并评价所得AhR：ARNT二聚体突变体的DNA结合能力，从而验证模型并选择最可靠的模型。这些发现为理解AhR功能活性的分子机制开辟了新的研究方向。
The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR. Computational modeling combined with experimental validation may give insight into structural and functional properties of protein systems. The basic Helix-Loop-Helix PER-ARNT-SIM (bHLH-PAS) proteins show conserved functional domains despite the broad range of functions exerted by the different systems. Within this protein family, the Aryl hydrocarbon Receptor (AhR) is known to mediate the toxic effects of a number of environmental contaminants, including dioxins and dioxin-like chemicals, and it also exerts other biochemical and physiological effects. Despite the absence of experimentally determined structures, theoretical models of the AhR PAS domains developed on the basis of homologous systems have allowed understanding of some aspects of the molecular mechanisms underlying its function. In this work we present alternative structural models of the transcriptionally active complex of AhR with the AhR Nuclear Translocator (ARNT) protein. Computational characterization of the modeled protein-protein interaction interfaces guided the design of mutagenesis experiments, and evaluation of the DNA binding ability of the resulting AhR:ARNT dimer mutants allowed validation of the models and selection of the most reliable one. These findings open new research directions for understanding the molecular mechanisms underlying the functional activity of the AhR.