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。生化数据得到了β结构域晶体结构的证实，我们与Susan Buchanan博士及其同事（NIDDK）合作解决了这一问题。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。EspP β结构域的晶体结构强烈表明，包含β结构域-过客结构域连接的短多肽片段位于由β结构域在过客结构域跨OM易位后形成的孔内。然而，我们发现，该片段在乘客结构域易位开始之前组装到β结构域中。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，我们的研究结果表明，整合的EspP β结构域到OM和易位的乘客域跨OM发生在一个单一的协同反应。有趣的是，最近的实验表明，该反应是由异源寡聚复合物（BAM复合物）介导的，该复合物先前已被证明可促进β桶蛋白整合到细菌OM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. The biochemical data were corroborated by the crystal structure of the beta domain, which we solved in collaboration with Dr. Susan Buchanan and co-workers (NIDDK). 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. The crystal structure of the EspP beta domain strongly suggested that a short polypeptide segment that encompasses the beta domain-passenger domain junction resides inside the pore formed by the beta domain following translocation of the passenger domain across the OM. We found, however, that this segment is assembled into the beta domain prior to the initiation of passenger domain translocation. 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 suggest 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. Interestingly, very recent experiments have suggested that this reaction is mediated by a heterooligomeric complex (Bam complex) that has previously been shown to promote the integration of beta barrel proteins into the bacterial OM.