Biogenesis of bacterial autotransporter proteins

在最近的实验中，我们一直使用大肠杆菌O157:H7产生的一种称为EspP的自转运蛋白作为模型蛋白来研究自转运蛋白的生物发生。我们一直在研究EspP乘客结构域跨OM易位的机制。使用Blue Native PAGE、分析超离心和其他生化方法，我们发现EspP β结构域表现为紧凑的单体，并形成一个太窄的通道，无法容纳折叠的多肽。令人惊讶的是，我们发现附着在EspP n端上的折叠蛋白结构域有效地跨OM转运，并且天然EspP客运结构域至少部分在外周质中折叠。这些看似矛盾的数据强烈表明，外部因素将客运结构域运输过OM，而β结构域的主要功能是将蛋白质靶向到OM。我们的研究结果挑战了自转运蛋白β结构域作为蛋白质转位酶的主流观点。我们还深入了解了EspP乘客结构域从细胞表面切割的机制。我们发现在一种新的自蛋白水解反应中释放了EspP乘客结构域。纯化后，未裂解的EspP前体在体外进行蛋白水解处理。蛋白质拓扑分析和突变研究强烈表明，该反应发生在β桶内，并揭示了两个保守残基，β结构域内的天冬氨酸（Asp1120）和裂解连接处P1位置的天冬氨酸（Asn1023）对于乘客结构域的裂解至关重要。有趣的是，这些残基对于两个远亲自身转运蛋白的蛋白水解过程也是必不可少的。这些数据强烈表明，Asp1120和Asn1023形成了一种不寻常的催化二联体，通过天冬酰胺的环化介导自裂解。值得注意的是，真核病毒衣壳成熟的机制也非常相似。我们提出的解理机制最近得到了EspP β结构域晶体结构的支持，我们与Buchanan实验室（LMB， NIDDK）合作解决了这个问题。结构表明β结构域形成了一个12股的β桶状结构，乘客/ β结构域解理连接点位于桶状孔内，大约位于OM的细胞外表面和质周表面之间。

In recent experiments we have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein to study autotransporter biogenesis. We have been examining the mechanism by which the EspP passenger domain is translocated across the OM. Using Blue Native PAGE, analytical ultracentrifugation and other biochemical methods we found that the EspP beta domain behaves as a compact monomer and forms a channel that is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain attached to the N-terminus of EspP is efficiently translocated across the OM and that the native EspP passenger domain folds at least partially in the periplasm. These apparently paradoxical data strongly suggest that an external factor transports the passenger domain across the OM and that the beta domain functions primarily to target the protein to the OM. Our results challenge the prevailing view of the autotransporter beta domain as a protein translocase. We have also obtained insights into the mechanism by which the EspP passenger domain is cleaved from the cell surface. We found that the EspP passenger domain is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor undergoes proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the beta barrel and revealed that two conserved residues, an aspartate within the beta domain (Asp1120) and an asparagine (Asn1023) at the P1 position of the cleavage junction are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp1120 and Asn1023 form an unusual catalytic dyad that mediates self-cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids. Our proposed cleavage mechanism was very recently supported by the crystal structure of the EspP beta domain, which we solved in collaboration with the Buchanan laboratory (LMB, NIDDK). The structure showed that the beta domain forms a 12-stranded beta-barrel with the passenger / beta domain cleavage junction located inside the barrel pore, approximately mid-way between the extracellular and periplasmic surfaces of the OM.