Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity.
Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity.
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DOI：
10.1016/j.str.2013.06.027
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发表时间：
2013-09-03
期刊：
影响因子：
--
通讯作者：
Hol WG
中科院分区：
文献类型：
--
作者：
Lu C;Turley S;Marionni ST;Park YJ;Lee KK;Patrick M;Shah R;Sandkvist M;Bush MF;Hol WG
II型分泌系统（T2 SS）是革兰氏阴性菌中跨越两层膜的多蛋白质机器，负责从周质分泌折叠蛋白质穿过外膜。到目前为止，关键的多结构域T2 SS组装ATP酶GspEEpsE在结构上还没有被表征为六聚体。在此，如天然质谱所示，当与作为辅助六聚体的Hcp 1融合时，获得了霍乱弧菌GspEEpsE的四个六聚体。GspEEpsE-Hcp 1融合体的酶活性比GspEEpsE单体的酶活性高约20倍，表明通过融合策略增加GspEEpsE的局部浓度是成功的。具有不同接头长度的GspEEpsE-Hcp 1融合物的晶体结构揭示了GspEEpsE的规则和细长的六聚体，其在亚基内的结构域取向和亚基组装中具有主要差异。对GspEEpsE-Hcp 1融合体的SAXS研究表明，GspEEpsE六聚体结构可能存在进一步的变异性。
The Type II Secretion System (T2SS), a multi-protein machinery spanning two membranes in Gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. The critical multi-domain T2SS assembly ATPase GspEEpsE had so far not been structurally characterized as a hexamer. Here, four hexamers of Vibrio cholerae GspEEpsE are obtained when fused to Hcp1 as an assistant hexamer, as shown by native mass spectrometry. The enzymatic activity of the GspEEpsE-Hcp1 fusions is ~20 times higher than that of a GspEEpsE monomer indicating that increasing the local concentration of GspEEpsE by the fusion strategy was successful. Crystal structures of GspEEpsE-Hcp1 fusions with different linker lengths reveal regular and elongated hexamers of GspEEpsE with major differences in domain orientation within subunits, and in subunit assembly. SAXS studies on GspEEpsE-Hcp1 fusions suggest that even further variability in GspEEpsE hexamer architecture is likely.