A solute-binding protein in the closed conformation induces ATP hydrolysis in a bacterial ATP-binding cassette transporter involved in the import of alginate
A solute-binding protein in the closed conformation induces ATP hydrolysis in a bacterial ATP-binding cassette transporter involved in the import of alginate
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DOI：
10.1074/jbc.m117.793992
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发表时间：
2017-09-22
影响因子：
4.8
通讯作者：
Hashimoto, Wataru
中科院分区：
文献类型：
--
作者：
Kaneko, Ai;Uenishi, Kasumi;Hashimoto, Wataru
革兰氏阴性菌鞘氨醇单胞菌A1通过细胞表面的小坑将藻酸盐结合到细胞中，而无需预先通过胞外酶解聚。藻酸盐跨细胞质膜的输入依赖于ATP结合盒转运体AlgM 1 M2 SS（AlgM 1、AlgM 2和AlgS的异源四聚体），其与周质溶质结合蛋白AlgQ 1或AlgQ 2合作;然而，AlgM 1 M2 SS介导的藻酸盐输入的几个细节尚不清楚。在此，我们分析了用具有不同聚合度（PD）的AlgQ 2和藻酸盐寡糖底物重构成脂质体后AlgM 1 M2 SS的ATP酶和转运活性。较长的藻酸盐寡糖（PD >= 5）刺激AlgM 1 M2 SS的ATP酶活性，但作为AlgM 1 M2 SS介导的转运的底物是惰性的，表明AlgM 1 M2 SS介导的ATP水解可以独立于底物转运而被刺激。使用X射线晶体学在AlgQ 2和长的海藻酸寡糖（PD 6-8）的存在下，并与潮湿的空气和胶水涂布的方法，我们确定了AlgM 1 M2 SS的晶体结构与寡糖结合AlgQ 2在3.6埃的分辨率。与非转运配体结合的周质solutebinding蛋白复合的ATP结合盒转运体的结构显示，AlgM 1 M2 SS和AlgQ 2分别采用内向和封闭构象。这些体外试验和结构分析表明，AlgM 1 M2 SS的内向构象和周质配体结合的AlgQ 2的封闭构象之间的相互作用诱导ATP水解的ATP结合蛋白AlgS。我们的结论是，底物结合的AlgQ 2在封闭的构象最初与AlgM 1 M2 SS相互作用，AlgM 1 M2 SS-AlgQ 2复合物，然后形成，这种形成是由ATP水解。
The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATPbinding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not wellunderstood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD >= 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6-8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 angstrom resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solutebinding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS-AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis.