Conformational Changes That Coordinate the Activity of BamA and BamD Allowing β-Barrel Assembly

Abstract

Most integral outer membrane proteins (OMPs) of Gram-negative bacteria, such as , assume a β-barrel structure. The β-barrel assembly machine (Bam), a five-member complex composed of β-barrel OMP BamA and four associated lipoproteins, BamB, BamC, BamD, and BamE, folds and inserts OMPs into the outer membrane. The two essential proteins BamA and BamD interact to stabilize two subcomplexes, BamAB and BamCDE, and genetic and structural evidence suggests that interactions between BamA and BamD occur via an electrostatic interaction between a conserved aspartate residue in a periplasmic domain of BamA and a conserved arginine in BamD. In this work, we characterize charge-change mutations at these key BamA and BamD residues and nearby charged residues in BamA with respect to OMP assembly and Bam complex stability. We show that Bam complex stability does not correlate with function, that BamA and BamD must adopt at least two active conformational states during OMP assembly, and that these charged residues are not required for function. Rather, these charged residues are important for coordinating the activities of BamA and BamD to allow efficient OMP assembly. We present a model of OMP assembly wherein recognition and binding of unfolded OMP substrate by BamA and BamD induce a signaling interaction between the two proteins, causing conformational changes necessary for the assembly reaction to proceed. By analogy to signal sequence recognition by SecYEG, we believe these BamA-BamD interactions ensure that both substrate and complex are competent for OMP assembly before the assembly reaction commences. Conformational changes in the proteins of the β-barrel assembly machine (Bam complex) are associated with the folding and assembly of outer membrane proteins (OMPs) in Gram-negative bacteria. We show that electrostatic interactions between the two essential proteins BamA and BamD coordinate conformational changes upon binding of unfolded substrate that allow the assembly reaction to proceed. Mutations affecting this interaction are lethal not because they destabilize the Bam complex but rather because they disrupt this coordination. Our model of BamA-BamD interactions regulating conformation in response to proper substrate interaction is reminiscent of conformational changes the secretory (Sec) machinery undergoes after signal sequence recognition that ensure protein quality control.

A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier

“…The bamA E373K mutation disrupts the interaction between BamA and BamD and is lethal in haploid due to a lack of proper coordination between the 2 proteins (41). This lethal phenotype can be suppressed by both intragenic suppressors in bamA (bamA K351E ) and extragenic suppressors in bamD (bamD R197L ) that bypass the need for proper coordination without restoring proper interaction between the 2 proteins (40,41). In our genetic characterization of bamA E470K , we discovered that this mutation is an intragenic suppressor of bamA E373K : a strain carrying bamA E373K/E470K in haploid is viable, although sick (Fig.…”

“…It is well established that BamA and BamD exist in multiple states and that OMP assembly requires these conformations to be properly coordinated. Proper interaction between the BamCDE and BamAB subcomplexes is necessary for each to be activated to fold and insert the OMP into the OM (39,40). Mutations that alter the conformation of either BamA or BamD can interfere with this coordination and prevent OMP assembly (41).…”

“…Mutations that alter the conformation of either BamA or BamD can interfere with this coordination and prevent OMP assembly (41). Suppressor mutations in bamA or bamD bypass the need for this coordination and restore OMP assembly (40,41). Since the bamA E470K mutation allows for retained function despite the presence of MRL-494 ( Fig.…”

“…Activation of the BAM complex involves communication between BamA and BamD, resulting in complementary conformational states that allow OMP assembly to proceed (32,39,40). Previous studies have isolated activating mutations in both BamA and BamD that suppress assembly-defective conditions and bypass the requirement for conformational coordination between these 2 essential proteins (38,39,41).…”

“…Previous studies have isolated activating mutations in both BamA and BamD that suppress assembly-defective conditions and bypass the requirement for conformational coordination between these 2 essential proteins (38,39,41). For instance, a bamA E373K mutation, which disrupts the essential communication between BamA and BamD (6), can be suppressed by activating mutations in BamA or BamD (40,41).…”