Erbel Paul J A, Card Paul B, Karakuzu Ozgur, Bruick Richard K, Gardner Kevin H
Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15504-9. doi: 10.1073/pnas.2533374100. Epub 2003 Dec 10.
Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFalpha and ARNT. Although this complex involves protein-protein interactions mediated by basic helix-loop-helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2alpha by NMR spectroscopy. We demonstrate that HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this beta-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.
生物体对氧可利用性的反应在发育、生理稳态及许多疾病过程中发挥着重要作用。在哺乳动物细胞中，这种适应性部分是由一条以缺氧诱导因子（HIF）为核心的保守途径介导的。HIF是一种异源二聚体蛋白复合物，由转录激活因子基本螺旋-环-螺旋Per-ARNT-Sim（PAS）（ARNT，芳烃受体核转运蛋白）结构域家族的两个成员HIFα和ARNT组成。尽管这种复合物涉及两种蛋白质中由基本螺旋-环-螺旋和PAS结构域介导的蛋白质-蛋白质相互作用，但对PAS结构域所起的作用了解甚少。为了解决这个问题，我们通过核磁共振波谱研究了人HIF-2α C末端PAS结构域的结构和相互作用。我们证明HIF-2α PAS-B在体外能结合类似的ARNT结构域，表明参与这种相互作用的残基位于HIF-2α中央β折叠的溶剂暴露侧。在这个表面突变残基不仅会破坏体外分离的PAS结构域之间的相互作用，还会干扰全长HIF在活细胞中对缺氧作出反应的能力。将我们的研究结果扩展到其他PAS结构域，我们发现这种β折叠界面广泛用于分子内和分子间相互作用，这为许多类型含PAS信号蛋白的特异性和调控提供了基础。
