Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
J Mol Biol. 2013 Mar 11;425(5):886-901. doi: 10.1016/j.jmb.2012.12.011. Epub 2012 Dec 26.
Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per-Arnt-Sim) and poly-HAMP (histidine kinase-adenylyl cyclase-methyl-accepting chemotaxis protein-phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iβ strand in a manner predicted to alter PAS-PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS-HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.
细菌受体通常具有模块化结构，包含不同功能的结构域，这些结构域组合在一起，对刺激产生信号。尽管在许多情况下已经研究了单个组件的特性，但关于不同模块集如何在全长受体中协同工作的信息却很少。在这里，我们研究了 Aer2 的结构，Aer2 是一种可溶性气体感应受体，已成为 PAS（Per-Arnt-Sim）和多-HAMP（组氨酸激酶-腺苷酸环化酶-甲基接受趋化蛋白-磷酸酶）结构域信号的模型。与先前确定的氰化物结合状态相比，血红素结合 PAS 结构域在铁离子、无配体状态下的晶体结构确定了配体结合诱导的构象变化，这些变化可能对信号机制至关重要。血红素口袋的变化与基于血红素的 PAS 传感器 FixL 和 EcDOS 有些相似，但以一种预测会改变 PAS-PAS 关联和全长 Aer2 中下游 HAMP 连接的方式传播到 Iβ 链。越来越复杂的 PAS 和多-HAMP 结构域片段的小角度 X 射线散射允许明确的结构域分配，并揭示了线性四级结构。Aer2 PAS 二聚体晶体结构很好地符合从头算小角度 X 射线散射分子包络，并且 PAS 之间的脉冲偶极 ESR 测量证实了 Aer2 中晶体学 PAS 排列。光谱和下拉测定均未能检测到 PAS 和 HAMP 结构域之间的直接相互作用。总体而言，Aer2 的信号机制与大肠杆菌 Aer 范例不同，其中侧 PAS-HAMP 接触是关键。我们提出了一种用于 Aer2 信号的直线模型，其中配体结合诱导 PAS 结构域结构和亚基缔合的改变，该改变通过多-HAMP 连接传递到下游结构域。
