Involvement of a conserved GFG motif region in substrate binding by RseP, an Escherichia coli S2P protease
Involvement of a conserved GFG motif region in substrate binding by RseP, an Escherichia coli S2P protease
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DOI：
10.1111/mmi.13659
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发表时间：
2017-06-01
影响因子：
3.6
通讯作者：
Akiyama, Yoshinori
中科院分区：
文献类型：
--
作者：
Akiyama, Koichiro;Hizukuri, Yohei;Akiyama, Yoshinori
RseP是大肠杆菌S2P家族的一种膜内裂解蛋白酶，通过对底物的特异性裂解参与胞质外应激反应的调控和膜质量的控制。最近的研究表明，PDZ结构域和MRE β环（膜可重入β环）参与底物识别；前者可用于防止具有大质周结构域的底物的切割，而后者可直接与底物的跨膜段相互作用并诱导其构象变化。然而，RseP特异性底物识别和切割的机制尚不完全清楚。本文研究了RseP第一胞质环区（C1N） n端部分包含高度保守的GFG基序的作用。Cys可修饰性实验表明，C1N部分像MRE β环一样插入膜。GFG基序区域的Pro（而非Cys）取代损害了RseP的蛋白水解功能，这表明该基序区域的高阶结构的重要性。多项证据表明，GFG基序区域直接与底物相互作用，也有助于参与RseP识别底物的MRE β环的功能。这些发现为S2P蛋白酶的底物识别机制提供了新的见解。
RseP, an Escherichia coli S2P family intramembrane cleaving protease, is involved in regulation of the extracytoplasmic stress response and membrane quality control through specific cleavage of substrates. Recent research suggested that the PDZ domains and the MRE beta-loop (membrane-reentrant beta-loop) are involved in substrate discrimination; the former would serve to prevent cleavage of substrates with a large periplasmic domain, whereas the latter would directly interact with the substrate's transmembrane segment and induce its conformational change. However, the mechanisms underlying specific substrate recognition and cleavage by RseP are not fully understood. Here, the roles of the N-terminal part of the first cytoplasmic loop region (C1N) of RseP that contains a highly conserved GFG motif were investigated. A Cys modifiability assay suggested that C1N is partly membrane-inserted like the MRE beta-loop. Pro, but not Cys, substitutions in the GFG motif region compromised the proteolytic function of RseP, suggesting the importance of a higher order structure of this motif region. Several lines of evidence indicated that the GFG motif region directly interacts with the substrate and also aids the function of the MRE beta-loop that participates in substrate recognition by RseP. These findings provide insights into the substrate recognition mechanisms of S2P proteases.