Assembly Factor Omp85 Recognizes Its Outer Membrane Protein Substrates by a Species-Specific C-Terminal Motif

TLDR

Integral β‑barrel proteins are found in the outer membranes of Gram‑negative bacteria, mitochondria, and chloroplasts, and their assembly requires a proteinaceous apparatus in which Omp85 is a conserved central component. The study aimed to elucidate the molecular mechanism of Omp85 by producing it from *E. coli* in inclusion bodies and refolding it in vitro. Omp85’s interaction with substrate proteins was examined in lipid‑bilayer experiments where it formed channels.

Abstract

Integral β-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence.