PDF(Pubmed)
Abstract：
Bacteria express lytic enzymes such as glycosidases, which have potentially self-destructive peptidoglycan (PG)-degrading activity and, therefore, require careful regulation in bacteria. The PG glycosidase EtgA is regulated by localization to the assembling type III secretion system (T3SS), generating a hole in the PG layer for the T3SS to reach the outer membrane. The EtgA localization was found to be mediated via EtgA interacting with the T3SS inner rod protein EscI. To gain structural insights into the EtgA recognition of EscI, we determined the 2.01 Å resolution structure of an EscI (51-87)-linker-EtgA fusion protein designed based on AlphaFold2 predictions. The structure revealed EscI residues 72-87 forming an α-helix interacting with the backside of EtgA, distant from the active site. EscI residues 56-71 also were found to interact with EtgA, with these residues stretching across the EtgA surface. The ability of the EscI to interact with EtgA was also probed using an EscI peptide. The EscI peptide comprising residues 66-87, slightly larger than the observed EscI α-helix, was shown to bind to EtgA using microscale thermophoresis and thermal shift differential scanning fluorimetry. The EscI peptide also had a two-fold activity-enhancing effect on EtgA, whereas the EscI-EtgA fusion protein enhanced activity over four-fold compared to EtgA. Our studies suggest that EtgA regulation by EscI could be trifold involving protein localization, protein activation, and protein stabilization components. Analysis of the sequence conservation of the EscI EtgA interface residues suggested a possible conservation of such regulation for related proteins from different bacteria.
摘要：
细菌表达裂解酶，如糖苷酶，具有潜在的自我破坏性肽聚糖（PG）降解活性，因此,需要小心调节细菌。PG糖苷酶EtgA通过定位到组装III型分泌系统(T3SS)来调节,在PG层中产生用于T3SS到达外膜的孔。发现EtgA定位是通过EtgA与T3SS内杆蛋白EscI相互作用介导的。为了获得对EtgA对EscI的认识的结构见解，我们确定了基于AlphaFold2预测设计的EscI（51-87）-接头-EtgA融合蛋白的2.01µ分辨率结构。该结构显示EscI残基72-87形成与EtgA背面相互作用的α-螺旋，远离活跃的地方。还发现EscI残基56-71与EtgA相互作用，这些残留物延伸穿过EtgA表面。还使用EscI肽探测了EscI与EtgA相互作用的能力。包含残基66-87的EscI肽，略大于观察到的EscIα-螺旋，使用微尺度热泳和热位移差示扫描荧光分析法显示与EtgA结合。EscI肽还对EtgA具有两倍的活性增强作用，而EscI-EtgA融合蛋白与EtgA相比活性提高了4倍。我们的研究表明，EscI对EtgA的调节可能是涉及蛋白质定位的三倍，蛋白质激活，和蛋白质稳定成分。对EscIEtgA界面残基的序列保守性的分析提示了对来自不同细菌的相关蛋白质的这种调节的可能保守性。
