大肠杆菌ATP依赖性ClpYQ蛋白酶由ClpYATPase/unfoldase和ClpQ肽酶组成。ClpY-六聚体中心孔I位点内的Tyr91残基对于将底物解折叠和转运到ClpQ的催化位点是重要的。我们已经确定了SulA的Degron位点(GFIMRP147),一种由ClpYQ识别的细胞分裂抑制剂,并且degron位点的Phe143残基是SulA天然折叠结构所必需的。然而,此degron位点与ClpYQ降解剂的功能关联未知。这里,我们研究了ClpYQ蛋白酶对底物识别和区分的分子见解。我们发现点突变体ClpYY91FQ,ClpYY91HQ,和ClpYY91WQ,在ClpY的第91个残基上带有环状结构,有效地降解了它们的天然底物,细菌甲烷磺酸盐(MMS)敏感性被抑制,cpsB::lacZ的β-半乳糖苷酶活性降低,和最低量的MBP-SulA在体内和体外降解分析。或者,模仿野生型SulA,SulAF143H,SulAF143K和SulAF143W,在SulA的第143个残基中具有环结构或阳离子侧基,在细菌细胞中被ClpYQ有效降解,还揭示了在下拉测定中在41°C下更短的半衰期和对ClpY的更高的结合亲和力。最后,ClpYY91FQ和ClpYY91HQ,能够有效降解SulAF143H和SulAF143K,突出了SulA第143和ClpY第91个残基之间的相应功能相互作用。根据可互换的取代氨基酸,我们的结果独特地表明,SulA143和ClpY91残基之间的瞬时π-π或阳离子-π相互作用可以适当地夹在底物的degron位点和蛋白酶(降解剂)的孔位点之间,用于底物识别和区分持续降解。
The Escherichia coli ATP-dependent ClpYQ protease constitutes ClpY ATPase/unfoldase and ClpQ peptidase. The Tyr91st residue within the central pore-I site of ClpY-hexamer is important for unfolding and translocating substrates into the catalytic site of ClpQ. We have identified the
degron site (GFIMRP147th) of SulA, a cell-division inhibitor recognized by ClpYQ and that the Phe143rd residue in
degron site is necessary for SulA native folded structure. However, the functional association of this
degron site with the ClpYQ degrader is unknown. Here, we investigated the molecular insights into substrate recognition and discrimination by the ClpYQ protease. We found that the point mutants ClpYY91FQ, ClpYY91HQ, and ClpYY91WQ, carrying a ring structure at the 91st residue of ClpY, efficiently degraded their natural substrates, evidenced by the suppressed bacterial methyl-methane-sulfonate (MMS) sensitivity, the reduced β-galactosidase activity of cpsB::lacZ, and the lowest amounts of MBP-SulA in both in vivo and in vitro degradation analyses. Alternatively, mimicking the wild-type SulA, SulAF143H, SulAF143K and SulAF143W, harboring a ring structure or a cation side-group in 143rd residue of SulA, were efficiently degraded by ClpYQ in the bacterial cells, also revealing shorter half-lives at 41 °C and higher binding affinities towards ClpY in pull-down assays. Finally, ClpYY91FQ and ClpYY91HQ, were capable of effectively degrading SulAF143H and SulAF143K, highlighting a correspondingly functional interaction between the SulA 143rd and ClpY 91st residues. According to the interchangeable substituted amino acids, our results uniquely indicate that a transient π-π or cation-π interaction between the SulA 143rd and ClpY 91st residues could be aptly gripped between the
degron site of substrates and the pore site of proteases (degraders) for substrate recognition and discrimination of the processive degradation.