Selective Pressure for Rapid Membrane Integration Constrains the Sequence of Bacterial Outer Membrane Proteins

The Moon-Fleming hydrophobicity scale suggests that the small thermodynamic penalty for inserting a basic residue into the lipid bilayer of the bacterial outer membrane (OM) does not account for the extreme evolutionary selection against arginine residues on the lipid-facing surfaces of OM proteins (OMPs). To explain this phenomenon, we investigated the consequences of introducing lipid-facing arginine residues into the β-barrel of two E. coli OMPs, an autotransporter (EspP) and outer membrane phospholipase A (OmpLA). We found that lipid-facing arginine residues do not affect the targeting of EspP to the Bam complex, an essential hetero-oligomer that catalyzes the membrane integration of OMPs, but markedly slow the membrane integration of the β-barrel domain. OmpLA mutants that contain lipid-facing arginine residues were likewise slowly incorporated into the OM in a strain that lacks the periplasmic protease DegP. In contrast to the EspP variants, however, the OmpLA mutants were degraded before they were inserted into the OM in a wild-type strain. The OmpLA arginine variants also folded slowly into large unilamellar vesicles in vitro. Taken together, the results suggest that lipid-facing arginine residues are strongly disfavored because proteins that assemble slowly are captured by the periplasmic quality control system through a kinetic partitioning mechanism or form prolonged interactions with the Bam complex that are potentially deleterious. Our results not only explain a key constraint on the sequence of OMPs, but also reveal important differences in the early stages of assembly of distinct classes of these proteins.