The Assembly of the Inverse Autotransporter Protein YeeJ is Driven by its C-terminal ß-strand.

ABSTRACT

Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a ß-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the ß1- and the ß12-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the ß1-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the ß1-strand with the ß3-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the ß12-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the ß-signals of classical autotransporter proteins and outer membrane ß-barrel proteins in general. We therefore propose that the ß12-strand of YeeJ acts as its ß-signal and that the assembly of the YeeJ ß-barrel is driven by its C-terminal ß-strand, like in most other autotransporter proteins, despite its inverted domain organization.