Bacterial Outer Membrane Proteins Are Targeted to the Bam Complex by Two Parallel Mechanisms

Abstract

Membrane proteins that are integrated into the outer membrane of Gram-negative bacteria typically contain a unique “β barrel” structure that serves as a membrane spanning segment. A conserved “β signal” motif is located at the C terminus of the β barrel of many outer membrane proteins (OMPs), but the function of this sequence is unclear. We found that mutations in the β signal slightly delayed the assembly of three model Escherichia coli OMPs by reducing their affinity for the barrel assembly machinery (Bam) complex, a heterooligomer that catalyzes β barrel insertion, and led to the degradation of a fraction of the protein in the periplasm. Interestingly, the absence of the periplasmic chaperone SurA amplified the effect of the mutations and caused the complete degradation of the mutant proteins. In contrast, the absence of another periplasmic chaperone (Skp) suppressed the effect of the mutations and considerably enhanced the efficiency of assembly. Our results reveal the existence of two parallel OMP targeting mechanisms that rely on a cis-acting peptide (the β signal) and a trans-acting factor (SurA), respectively. Our results also challenge the long-standing view that periplasmic chaperones are redundant and provide evidence that they have specialized functions. IMPORTANCE Proteins that are embedded in the outer membrane of Gram-negative bacteria (OMPs) play an important role in protecting the cell from harmful chemicals. OMPs share a common architecture and often contain a conserved sequence motif (β motif) of unknown function. Although OMPs are escorted to the outer membrane by proteins called chaperones, the exact function of the chaperones is also unclear. Here, we show that the β motif and the chaperone SurA both target OMPs to the β barrel insertion machinery in the outer membrane. In contrast, the chaperone Skp delivers unintegrated OMPs to protein degradation complexes. Our results challenge the long-standing view that chaperones are functionally redundant and strongly suggest that they have specialized roles in OMP targeting and quality control.

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

“…Nevertheless, the β-signal binding pocket is not totally filled. Presumably this property enables BAM to accommodate the less common subset of OMPs that have β-signals terminated by tryptophan or tyrosine instead of phenylalanine (Struyve et al, 1991; Wang et al, 2021). The conserved β-signal residue at the -3 position of EspP (Y1298) interacts with BamD L124 and is oriented into the membrane plane at a depth corresponding to the aromatic girdles of fully folded canonical β-barrels (Figure 1F).…”

“…However, the BamA-β-signal interaction constitutes the first observation of an inter-barrel mortise-tenon-like joint. The binding of BAM to the β-signal provides an explanation for the finding that mutations of the terminal aromatic residue cause severe assembly defects and lead to OMP degradation in vivo (Gessmann et al, 2014; Lee et al, 2018; Wang et al, 2021). Presumably the mutations reduce the binding affinity of incoming OMPs to BAM, prevent their progression to the hybrid-barrel stage, and result in exposure to periplasmic proteases.…”

“…Transmembrane β-barrels are also stabilized in the OM by parallel “girdles” of membrane-facing aromatic residues (Schulz, 2000). The majority (77%) of β-barrels also have a highly conserved C-terminal motif called the “β-signal” that contains an essential terminal phenylalanine residue of unknown function (Struyve et al, 1991; Wang et al, 2021). In bacteria, the assembly (folding and integration) of β- b arrels is catalyzed by a heterooligomer called the β- b arrel a ssembly m achine (BAM) (Heinz and Lithgow, 2014; Heinz et al, 2015; Wu et al, 2005).…”

“…Here, we examined the folding of a model E. coli O157:H7 OMP (EspP) that contains a stably closed β-seam, an average sized β-barrel (12 β-strands), and a canonical β-signal (Barnard et al, 2007; Franklin et al, 2018; Wang et al, 2021). By using single-particle cryo-EM to analyze an assembly-arrested form of the protein associated with BAM in native-nanodiscs that contain components directly extracted from the bacterial OM (unlike previous structural studies that analyzed BAM in detergent or nanodiscs with synthetic phospholipid bilayers), we were able to visualize multiple intermediate stages of β-barrel folding.…”

How do Chaperones Bind (Partly) Unfolded Client Proteins?

“…The bacterial periplasmic chaperones Skp (Seventeen Kilodalton Protein; Chen and Henning (1996) ) and SurA [Survival factor A, Behrens et al (2001) , Bitto and McKay (2002) ] share the pool of OMP clients; initially, they appeared to have redundant function in escorting the OMPs to the BAM complex of the outer membrane [ Sklar et al (2007) , McMorran et al (2015) ]. Recent work Wang et al (2021) indicates that the SurA chaperone, with the PPIase (peptidyl-prolyl cis -trans isomerase) activity Sklar et al (2007) , has a role in targeting OMPs to the BAM complex, while the chaperone Skp delivers unintegrated OMPs to the DegP for their degradation. The client proteins that have been extensively used as models for interactions, OmpX and OmpA, share similar hydrophobic and polar properties of their amino-acid sequence in the transmembrane parts ( Figures 4A,B ); OmpA has an additional soluble periplasmic domain on the C-terminus (but several studies used only the TM part as client).…”