Biogenesis of bacterial autotransporter proteins

我们一直使用大肠杆菌O157：H7产生的自体转运蛋白ESPP作为模型蛋白来研究自体转运蛋白的生物发生。在一项调查中，我们一直在研究ESPP乘客域跨OM转移的机制。使用几种不同的生化方法，我们发现ESPPβ结构域表现为一个紧凑的单体，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，我们发现连接到ESPP N-末端的折叠蛋白结构域有效地跨OM易位，并且天然的ESPP乘客结构域至少部分在周质中折叠。这些明显自相矛盾的数据有力地表明，一个外部因素将客体结构域穿过OM，而β结构域的功能主要是将蛋白质靶向OM。我们的结果挑战了流行的观点，即自转运体β结构域起蛋白质转位酶的作用。在第二条线的调查中，我们已经深入了解了ESPP客体结构域从细胞表面被切割的机制。我们发现，ESPP的客体结构域在一种新的自蛋白分解反应中被释放。纯化后，对未切割的ESPP前体进行体外蛋白降解处理。蛋白质拓扑分析和突变研究有力地表明，该反应发生在β桶内，并揭示了两个保守残基，即β结构域中的天冬氨酸(Asp1120)和位于裂解连接P1位的天冬酰胺(Asn1023)对Passenger结构域的切割是必不可少的。有趣的是，这些残基对两个远亲自体转运蛋白的蛋白质分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聚体，通过天冬酰胺的环化来介导自我切割。值得注意的是，对于真核病毒衣壳的成熟，已经提出了一个非常相似的机制。我们提出的切割机制得到了ESPPβ结构域晶体结构的支持，我们与Susan Buchanan博士和她的同事(LMB，NIDDK)合作解决了这一问题。结构表明，β结构域形成一个12链的β结构域，乘客/β结构域裂解连接位于管孔内，大约在OM的细胞外表面和周质表面之间的中间。在最近的研究中，我们表明，包含切割连接的多肽片段在乘客结构域跨OM移位之前组装到β桶中。这些数据强烈表明，ESPP的β结构域和嵌入的多肽片段作为单一的预形成单位整合到OM中。综上所述，我们的结果提出了这样一种可能性，即ESPPβ结构域整合到OM中，以及乘客结构域跨OM的移位发生在单一的协同反应中。

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.