Biogenesis of bacterial autotransporter proteins

我们一直在使用由E. coli O 157：H7中的EspP蛋白作为研究自转运蛋白生物合成的模型蛋白。 在一条调查线，我们一直在研究的机制，其中EspP乘客域是跨OM易位。 使用几种不同的生物化学方法，我们发现EspP β结构域表现为紧凑的单体，并形成太窄而不能容纳折叠多肽的通道。 令人惊讶的是，我们发现连接到EspP的N-末端的折叠的蛋白质结构域有效地跨OM易位，并且天然EspP乘客结构域至少部分地在周质中折叠。 这些明显自相矛盾的数据强烈表明，一个外部因素运输乘客域跨OM和β结构域的功能主要是针对OM的蛋白质。我们的研究结果挑战了自转运蛋白β结构域作为蛋白质移位酶的流行观点。 在第二条调查线中，我们已经深入了解了EspP乘客结构域从细胞表面裂解的机制。 我们发现EspP乘客结构域在一种新的自蛋白水解反应中释放。 纯化后，未切割的EspP前体在体外经历蛋白水解加工。 蛋白质拓扑结构的分析以及突变研究强烈表明，反应发生在β桶内，并揭示了两个保守的残基，β结构域内的天冬氨酸（Asp 1120）和天冬酰胺（Asn 1023）在P1位置的切割交界处是必不可少的乘客域切割。 有趣的是，这些残基也是必不可少的两个远亲的autotorporters的蛋白水解加工。 这些数据强烈表明，Asp 1120和Asn 1023形成一个不寻常的催化二联体，介导通过环化的天冬酰胺的自我裂解。 值得注意的是，一个非常相似的机制已经提出了真核病毒衣壳的成熟。 我们提出的切割机制得到了EspP β结构域的晶体结构的支持，我们与Susan Buchanan博士及其同事（LMB，NIDDK）合作解决了这个问题。结构显示，β结构域形成一个12链β桶，其中乘客/β结构域切割接头位于桶孔内，大约位于OM的细胞外表面和周质表面之间的中间位置。在最近的研究中，我们表明，多肽片段，包括切割交界处组装成β桶之前，乘客域跨OM易位。 数据强烈表明EspP β结构域和嵌入的多肽片段作为单个预形成单元整合到OM中。 两者合计，我们的研究结果提出了一种可能性，即EspP β结构域整合到OM中和乘客结构域跨OM的易位发生在单个协同反应中。

We have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein to study autotransporter biogenesis. In one line of investigation we have been examining the mechanism by which the EspP passenger domain is translocated across the OM. Using several different 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 that the autotransporter beta domain functions as a protein translocase. In a second line of investigation we have obtained insight 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 supported by the crystal structure of the EspP beta domain, which we solved in collaboration with Dr. Susan Buchanan and her colleagues (LMB, NIDDK). The structure shows 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. In very recent studies we showed that the polypeptide segment that encompasses the cleavage junction is assembled into the beta barrel prior to the translocation of the passenger domain across the OM. The data strongly suggest that the EspP beta domain and an embedded polypeptide segment are integrated into the OM as a single pre-formed unit. Taken together, our results raise the possibility that the integration of the EspP beta domain into the OM and the translocation of the passenger domain across the OM occur in a single concerted reaction.