The role of calcium binding to the EF-hand-like motif in bacterial solute-binding protein for alginate import
The role of calcium binding to the EF-hand-like motif in bacterial solute-binding protein for alginate import
复制标题
钙与细菌溶质结合蛋白中 EF 手状基序结合对藻酸盐输入的作用
DOI：
10.1093/bbb/zbab170
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Hashimoto Wataru
中科院分区：
文献类型：
--
作者：
Okumura Kenji;Maruyama Yukie;Takase Ryuichi;Mikami Bunzo;Murata Kousaku;Hashimoto Wataru
革兰氏阴性鞘氨醇单胞菌A1通过细胞表面藻酸盐结合蛋白（AlgQ 2）依赖的ATP结合盒转运蛋白（AlgM 1 M2 SS）将酸性多糖藻酸盐掺入细胞质。我们通过在钙结合位点引入突变来研究AlgQ 2中EF-手样基序结合的钙的功能。AlgQ 2突变体（D179 A/E180 A）的X-射线晶体学显示缺乏钙结合和EF-手样基序的显著紊乱。与野生型AlgQ 2不同的是，突变体在菌株A1生长的温度下相当不稳定，尽管不饱和藻酸盐寡糖通过形成底物/蛋白质复合物来稳定突变体。在ATP酶和海藻酸钠转运实验中，野生型和突变型AlgQ 2在不饱和海藻酸钠四糖的存在下诱导了AlgM 1 M2 SS ATP酶活性。这些结果表明，结合到EF-手样基序的钙稳定了未结合底物的AlgQ 2，但对于结合底物的AlgQ 2和AlgM 1 M2 SS的络合不是必需的。
Gram-negativeSphingomonassp. A1 incorporates acidic polysaccharide alginate into the cytoplasm via a cell-surface alginate-binding protein (AlgQ2)-dependent ATP-binding cassette transporter (AlgM1M2SS). We investigated the function of calcium bound to the EF-hand-like motif in AlgQ2 by introducing mutations at the calcium-binding site. The X-ray crystallography of the AlgQ2 mutant (D179A/E180A) demonstrated the absence of calcium binding and significant disorder of the EF-hand-like motif. Distinct from the wild-type AlgQ2, the mutant was quite unstable at temperature of strain A1 growth, although unsaturated alginate oligosaccharides stabilized the mutant by formation of substrate/protein complex. In the assay of ATPase and alginate transport by AlgM1M2SS reconstructed in the liposome, the wild-type and mutant AlgQ2 induced AlgM1M2SS ATPase activity in the presence of unsaturated alginate tetrasaccharide. These results indicate that the calcium bound to EF-hand-like motif stabilizes the substrate-unbound AlgQ2 but is not required for the complexation of substrate-bound AlgQ2 and AlgM1M2SS.