Substrate recognition and binding by RseP, an Escherichia coli intramembrane protease
Substrate recognition and binding by RseP, an Escherichia coli intramembrane protease
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DOI：
10.1074/jbc.m709984200
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发表时间：
2008-04-11
影响因子：
4.8
通讯作者：
Akiyama, Yoshinori
中科院分区：
文献类型：
--
作者：
Koide, Kayo;Ito, Koreaki;Akiyama, Yoshinori
大肠杆菌RseP属于膜内切割蛋白酶的S2 P家族。RseP催化膜结合的抗σ（E）E蛋白RseA的蛋白水解裂解，作为σ（E）胞质外应激反应途径中跨膜信号转导的必要步骤。RseP切割膜蛋白的跨膜片段，但其底物识别和蛋白水解作用的分子机制在很大程度上仍然未知。在这里，我们分析了RseP和底物膜蛋白之间的相互作用。免疫共沉淀试验表明，螺旋不稳定的残基在底物跨膜段，这是以前被证明是所需的有效的蛋白水解的底物RseP，稳定的底物-RseP相互作用。RseP的第三跨膜区（TM 3）中的某些氨基酸残基（包括进化上保守的氨基酸残基）的取代减弱了RseP-底物相互作用。RseP TM 3和RseA跨膜片段中Cys取代的特定组合导致氧化后形成二硫键，表明RseP的TM 3直接结合底物。这些结果提供了深入了解的RseP的膜蛋白水解的机制。
Escherichia coli RseP belongs to the S2P family of intramembrane cleaving proteases. RseP catalyzes proteolytic cleavage of the membrane-bound anti-sigma(E) E protein RseA as an essential step in transmembrane signal transduction in the sigma(E) extracytoplasmic stress response pathway. RseP cleaves transmembrane segments of membrane proteins, but the molecular mechanisms of its substrate recognition and proteolytic action remain largely unknown. Here we analyzed interaction between RseP and substrate membrane proteins. Co-immunoprecipitation assays showed that helix-destabilizing residues in a substrate transmembrane segment, which were previously shown to be required for efficient proteolysis of the substrate by RseP, stabilize the substrate-RseP interaction. Substitutions of certain amino acid residues, including those evolutionarily conserved, in the third transmembrane region (TM3) of RseP weakened the RseP-substrate interaction. Specific combinations of Cys substitutions in RseP TM3 and in the RseA transmembrane segment led to the formation of disulfide bonds upon oxidation, suggesting that TM3 of RseP directly binds the substrate. These results provide insights into the mechanism of membrane protein proteolysis by RseP.