Gas Sensing and Signaling in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor
Gas Sensing and Signaling in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor
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DOI：
10.1128/jb.00003-17
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发表时间：
2017-09-01
影响因子：
3.2
通讯作者：
Watts, Kylie J.
中科院分区：
文献类型：
--
作者：
Garcia, Darysbel;Orillard, Emilie;Watts, Kylie J.
来自铜绿假单胞菌的 Aer2 化学感受器包含一个 PAS 传感结构域，可协调 b 型血红素和响应 O-2、CO 或 NO 结合的信号。 PAS-血红素结构表明，Aer2 通过 3(10) 螺旋上的 His 残基（E eta 上的 H234）独特地协调血红素，通过 Trp 残基 (W283) 稳定 O-2 结合，并通过 W283 和相邻的 Leu 残基 (L264) 发出信号。配体结合可能会取代 L264 并重新定向 W283 以与配体形成氢键。在这里，我们阐明了 Aer2-PAS 结合血红素、调节配体结合和启动构象信号传导的机制。 H234 协调血红素，但血红素裂隙中额外的疏水残基对于稳定的血红素结合也至关重要。 O2 似乎是天然的 Aer2 配体（解离常数 [K-d] 为 16 μM）。除了一个例外，结合 O-2 的突变体可以发出信号，而许多结合 CO 的突变体却不能。 W283 稳定 O-2 结合但不稳定 CO 结合，并且它是信号启动所必需的；无法稳定 O-2 的 W283 突变体被快速氧化为 Fe(III)。 W283F 是唯一能够以野生型亲和力结合 O-2 的色氨酸突变体。残基 264 的大小和性质对于气体结合和信号传导非常重要：L264W 阻断 O-2 结合，L264A 和 L264G 引起 O-2 介导的氧化，L264K 形成六配位血红素。我们的数据表明，当 O-2 与 Aer2 结合时，L264 与 W283 一起移动以启动构象信号。然后信号从 PAS 结构域传播，调节 C 端 HAMP 和激酶控制结构域，最终调节细胞反应。 重要性 铜绿假单胞菌是一种普遍存在的环境细菌和机会性病原体，可感染多个身体部位，包括囊性纤维化患者的肺部。铜绿假单胞菌通过四种化学感应系统感知环境并对其做出反应。其中三个系统调节生物膜形成、抽搐运动和趋化性。第四个系统 Che2 的作用尚不清楚，但与毒力有关。 Che2 系统包含一个称为 Aer2 的化学感受器，其中包含一个结合血红素并感知氧气的 PAS 传感结构域。在这里，我们展示了 Aer2 使用前所未有的机制来结合 O-2 并启动信号传导。这些研究首次从结构上证实了先前提出的 Aer2-PAS 信号传导机制以及 Aer2-PAS 受体的信号传导模型。
The Aer2 chemoreceptor from Pseudomonas aeruginosa contains a PAS sensing domain that coordinates b-type heme and signals in response to the binding of O-2, CO, or NO. PAS-heme structures suggest that Aer2 uniquely coordinates heme via a His residue on a 3(10) helix (H234 on E eta), stabilizes O-2 binding via a Trp residue (W283), and signals via both W283 and an adjacent Leu residue (L264). Ligand binding may displace L264 and reorient W283 for hydrogen bonding to the ligand. Here, we clarified the mechanisms by which Aer2-PAS binds heme, regulates ligand binding, and initiates conformational signaling. H234 coordinated heme, but additional hydrophobic residues in the heme cleft were also critical for stable heme binding. O2 appeared to be the native Aer2 ligand (dissociation constant [K-d] of 16 mu M). With one exception, mutants that bound O-2 could signal, whereas many mutants that bound CO could not. W283 stabilized O-2 binding but not CO binding, and it was required for signal initiation; W283 mutants that could not stabilize O-2 were rapidly oxidized to Fe(III). W283F was the only Trp mutant that bound O-2 with wildtype affinity. The size and nature of residue 264 was important for gas binding and signaling: L264W blocked O-2 binding, L264A and L264G caused O-2-mediated oxidation, and L264K formed a hexacoordinate heme. Our data suggest that when O-2 binds to Aer2, L264 moves concomitantly with W283 to initiate the conformational signal. The signal then propagates from the PAS domain to regulate the C-terminal HAMP and kinase control domains, ultimately modulating a cellular response.IMPORTANCE Pseudomonas aeruginosa is a ubiquitous environmental bacterium and opportunistic pathogen that infects multiple body sites, including the lungs of cystic fibrosis patients. P. aeruginosa senses and responds to its environment via four chemosensory systems. Three of these systems regulate biofilm formation, twitching motility, and chemotaxis. The role of the fourth system, Che2, is unclear but has been implicated in virulence. The Che2 system contains a chemoreceptor called Aer2, which contains a PAS sensing domain that binds heme and senses oxygen. Here, we show that Aer2 uses unprecedented mechanisms to bind O-2 and initiate signaling. These studies provide both the first functional corroboration of the Aer2-PAS signaling mechanism previously proposed from structure as well as a signaling model for Aer2-PAS receptors.