SspB delivery of substrates for ClpXP proteolysis probed by the design of improved degradation tags
SspB delivery of substrates for ClpXP proteolysis probed by the design of improved degradation tags
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DOI：
10.1073/pnas.0404733101
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发表时间：
2004-08-17
影响因子：
11.1
通讯作者：
Sauer, RT
中科院分区：
文献类型：
--
作者：
Hersch, GL;Baker, TA;Sauer, RT
ssrA-降解标签序列含有SspB衔接子和ClpXP蛋白酶的ClpX组分的连续结合位点。虽然SspB通常增强ClpXP降解ssrA标记的底物，它抑制蛋白水解的条件下，防止拴系到ClpX。通过增加ssrA标签中蛋白酶和衔接子结合决定簇之间的间距，获得了显示出改善的SspB介导的与ClpXP的结合和ClpXP降解的底物。这些延伸标签底物也显示出显著降低的条件性抑制，但正常结合SspB。野生型和突变体标签都表现出高度动态的SspB相互作用。总之，这些结果强烈支持SspB和ClpX同时结合到ssrA标签的递送模型，但也表明SspB和ClpX之间的冲突削弱了同时结合。在底物递送期间，通过将SspB拴系到ClpX来克服这种信号掩蔽，这确保局部浓度足够高以驱动标签接合。这种先阻碍后刺激的机制可能已经进化到允许额外的调节水平，并且可能是衔接子介导的蛋白质降解的共同特征。
The ssrA-degradation tag sequence contains contiguous binding sites for the SspB adaptor and the ClpX component of the ClpXP protease. Although SspB normally enhances ClpXP degradation of ssrA-tagged substrates, it inhibits proteolysis under conditions that prevent tethering to ClpX. By increasing the spacing between the protease and adaptor-binding determinants in the ssrA tag, substrates were obtained that displayed improved SspB-mediated binding to and degradation by ClpXP. These extended-tag substrates also showed significantly reduced conditional inhibition but bound SspB normally. Both wild-type and mutant tags showed highly dynamic SspB interactions. Together, these results strongly support delivery models in which SspB and ClpX bind concurrently to the ssrA tag, but also suggest that clashes between SspB and ClpX weaken simultaneous binding. During substrate delivery, this signal masking is overcome by tethering SspB to ClpX, which ensures local concentrations high enough to drive tag engagement. This obstruct-then-stimulate mechanism may have evolved to allow additional levels of regulation and could be a common trait of adaptor-mediated protein degradation.