Laboratory of Crystallographic Studies, IACT (CSIC-UGR), Granada, Spain.
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
FEBS J. 2021 Apr;288(7):2294-2310. doi: 10.1111/febs.15580. Epub 2020 Oct 27.
Signalling through chemosensory pathways is typically initiated by the binding of signal molecules to the chemoreceptor ligand binding domain (LBD). The PcaY_PP chemoreceptor from Pseudomonas putida KT2440 is characterized by an unusually broad signal range, and minimal requisites for signal binding are the presence of a C6-membered ring and that of a carboxyl group. Previous studies have shown that only some of the multiple signals recognized by this chemoreceptor are of apparent metabolic value. We report here high-resolution structures of PcaY_PP-LBD in the absence and presence of four cognate chemoeffectors and glycerol. The domain formed a four-helix bundle (4HB), and both ligand binding sites of the dimer were occupied with the high-affinity ligands protocatechuate and quinate, whereas the lower-affinity ligands benzoate and salicylate were present in only one site. Ligand binding was verified by microcalorimetric titration of site-directed mutants revealing important roles of an arginine and number of polar residues that establish an extensive hydrogen bonding network with bound ligands. The comparison of the apo and holo structures did not provide evidence for this receptor employing a transmembrane signalling mechanism that involves piston-like shifts of the final helix. Instead, ligand binding caused rigid-body scissoring movements of both monomers of the dimer. Comparisons with the 4HB domains of the Tar and Tsr chemoreceptors revealed significant structural differences. Importantly, the ligand binding site in PcaY_PP-LBD is approximately 8 Å removed from that of the Tar and Tsr receptors. Data indicate a significant amount of structural and functional diversity among 4HB domains. DATABASES: The coordinates and structure factors have been deposited in the protein data band with the following IDs: 6S1A (apo form), 6S18 (bound glycerol), 6S33 (bound protocatechuate), 6S38 (bound quinate), 6S3B (bound benzoate) and 6S37 (bound salicylate).
化学感应途径的信号传递通常是由信号分子与化学感受器配体结合域（LBD）结合引发的。恶臭假单胞菌 KT2440 的 PcaY_PP 化学感受器的特点是信号范围异常广泛，而信号结合的最小要求是存在一个 C6 元环和一个羧基。先前的研究表明，这种化学感受器识别的多种信号中，只有一些具有明显的代谢价值。我们在这里报告了 PcaY_PP-LBD 在没有和存在四种同源化学效应物和甘油的情况下的高分辨率结构。该结构域形成了一个四螺旋束（4HB），二聚体的两个配体结合位点都被高亲和力配体原儿茶酸和奎尼酸占据，而低亲和力配体苯甲酸和水杨酸仅存在于一个位点。通过微热量滴定对定点突变体的实验验证了配体结合，揭示了一个精氨酸和许多极性残基的重要作用，这些残基与结合的配体建立了广泛的氢键网络。对apo 和 holo 结构的比较没有提供证据表明该受体采用涉及最后一个螺旋活塞式位移的跨膜信号转导机制。相反，配体结合导致二聚体的两个单体刚性体剪式运动。与 Tar 和 Tsr 化学感受器的 4HB 结构域的比较显示出显著的结构差异。重要的是，PcaY_PP-LBD 的配体结合位点与 Tar 和 Tsr 受体的配体结合位点相差约 8 Å。数据表明 4HB 结构域之间存在大量的结构和功能多样性。数据库：坐标和结构因子已被提交到蛋白质数据银行，以下 ID 分别为：6S1A（apo 形式）、6S18（结合甘油）、6S33（结合原儿茶酸）、6S38（结合奎尼酸）、6S3B（结合苯甲酸）和 6S37（结合水杨酸）。
