Limited tolerance towards folded elements during secretion of the autotransporter Hbp
Limited tolerance towards folded elements during secretion of the autotransporter Hbp
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DOI：
10.1111/j.1365-2958.2007.05605.x
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发表时间：
2007-03-01
影响因子：
3.6
通讯作者：
Luirink, Joen
中科院分区：
文献类型：
--
作者：
Jong, Wouter S. P.;ten Hagen-Jongman, Corinne M.;Luirink, Joen
致病性革兰氏阴性菌分泌的许多毒力因子属于自转运蛋白（AT）家族。AT由乘客结构域和β结构域组成，乘客结构域是实际分泌的部分，β结构域促进乘客结构域跨外膜转移。在这里，我们分析了折叠和易位的AT乘客，使用大肠杆菌血红蛋白蛋白酶（Hbp）作为模型蛋白。双半胱氨酸诱变，煽动独特的晶体结构的Hbp乘客，导致分子内二硫键的形成依赖于周质酶DsbA。由二硫键连接的小环不干扰Hbp的分泌。相反，Hbp乘客的不同结构域之间的键完全阻断分泌，导致周质蛋白酶DegP降解。在缺乏DegP的情况下，转运中间体在外膜中积累。在将钙调蛋白折叠部分插入Hbp中时形成类似的堵塞中间体。这些数据表明，Hbp可以在周质中折叠，但必须保持一定程度的灵活性和/或适度的宽度，以允许跨外膜易位。
Many virulence factors secreted by pathogenic Gram-negative bacteria belong to the autotransporter (AT) family. ATs consist of a passenger domain, which is the actual secreted moiety, and a beta-domain that facilitates the transfer of the passenger domain across the outer membrane. Here, we analysed folding and translocation of the AT passenger, using Escherichia coli haemoglobin protease (Hbp) as a model protein. Dual cysteine mutagenesis, instigated by the unique crystal structure of the Hbp passenger, resulted in intramolecular disulphide bond formation dependent on the periplasmic enzyme DsbA. A small loop tied off by a disulphide bond did not interfere with secretion of Hbp. In contrast, a bond between different domains of the Hbp passenger completely blocked secretion resulting in degradation by the periplasmic protease DegP. In the absence of DegP, a translocation intermediate accumulated in the outer membrane. A similar jammed intermediate was formed upon insertion of a calmodulin folding moiety into Hbp. The data suggest that Hbp can fold in the periplasm but must retain a certain degree of flexibility and/or modest width to allow translocation across the outer membrane.