Abstract
ProOmpA is a preprotein that is translocated across the plasma membrane by the general secretory pathway in Escherichia coli. The molecular chaperon SecB in Sec pathway can recognize and bind proOmpA for its translocation. However, the structure of the SecB/proOmpA complex remains unknown. Here, we constructed an uncleavable proOmpA fused with metallothionein at its C-terminus and labeled it with metals in vitro for the study of cryo-electron microscopy. Using single particle cryo-electron microscopy, we reconstructed 3D structure of the stable SecB/proOmpA complex. The structure shows that the major portion of preprotein locates on one side of SecB tetramer, resulting in an asymmetric binding pattern. This work also provides a possible approach to the structure determination of small protein complexes by cryo-electron microscopy.
Highlights
The general secretory pathway (Sec pathway) is responsible for the translocation of a subset of proteins across the inner membrane to the periplasm in E. coli
The structure of the SecB/preprotein complex remains largely unclear, which limits our understanding of the molecular mechanism underlying how SecB interacts with preproteins
The complex of SecB and proOmpA is very small with the molecular weight of only,110 kDa, and the complex tended to form aggregates in vitro [19]. All of these make the structural study of the SecB/proOmpA complex by cryo-electron microscopy (EM) difficult
Summary
The general secretory pathway (Sec pathway) is responsible for the translocation of a subset of proteins across the inner membrane to the periplasm in E. coli. The signal peptide of proOmpA is cleaved by signal peptidase during the process of secretion, resulting in the formation of mature OmpA. This cleavage often makes the purified proOmpA contaminated with OmpA. The complex of SecB and proOmpA is very small with the molecular weight of only ,110 kDa, and the complex tended to form aggregates in vitro [19]. All of these make the structural study of the SecB/proOmpA complex by cryo-electron microscopy (EM) difficult. Only the 3D structure of the complex of SecB with OmpA was obtained by negative staining EM [19]
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