Crystal Structure of BamB from Pseudomonas aeruginosa and Functional Evaluation of Its Conserved Structural Features

Abstract

The assembly of β-barrel Outer Membrane Proteins (OMPs) in the outer membrane is essential for Gram-negative bacteria. The process requires the β-Barrel Assembly Machine (BAM), a multiprotein complex that, in E. coli, is composed of the OMP BamA and four lipoproteins BamB-E. Whereas BamA and BamD are essential, deletion of BamB, C or E produce membrane permeability defects. Here we present the high-resolution structure of BamB from Pseudomonas aeruginosa. This protein can complement the deletion of bamB in E. coli indicating that they are functionally equivalent. Conserved structural features include an eight-bladed β-propeller fold stabilized by tryptophan docking motifs with a central pore about 8 Å in diameter at the narrowest point. This pore distinguishes BamB from related β-propellers, such as quinoprotein dehydrogenases. However, a double mutation designed to block this pore was fully functional indicating that the opening is not essential. Two loops protruding from the bottom of the propeller are conserved and mediate binding to BamA. Conversely, an additional loop only present in E. coli BamB is not required for function. A cluster of highly conserved residues in a groove between blades 6 and 7 is crucial for proper BamB folding or biogenesis. It has been proposed that BamB may bind nascent OMPs by β-augmentation to its propeller outer strands, or recognize the aromatic residue signature at the C-terminus of OMPs. However, Isothermal Titration Calorimetry experiments and structural analysis do not support these proposals. The structural and mutagenesis analysis suggests that the main function of BamB is to bind and modulate BamA, rather than directly interact with nascent OMPs.

The Structure of a BamA-BamD Fusion Illuminates the Architecture of the β-Barrel Assembly Machine Core

“…Based on analysis of its high-resolution structure, BamB has been proposed to interact directly with nascent OMPs (Gatsos et al, 2008; Heuck et al, 2011). However, experimental testing of those proposals has been negative (Jansen et al, 2012). The non-essential lipoprotein may instead have a role in modulating the conformations of the essential subunits BamA and BamD.…”

“…The structures of all the individual BAM subunits have been reported (Albrecht and Zeth, 2011; Endo et al, 2011; Heuck et al, 2011; Jansen et al, 2012; Kim and Paetzel, 2011; Knowles et al, 2011; Noinaj et al, 2011; Noinaj et al, 2013; Sandoval et al, 2011; Warner et al, 2011). BamA is a β-barrel OMP with an N-terminal periplasmic domain composed of five po lypeptide tr anslocation a ssociated (POTRA) motifs.…”

Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA, the Central Component of the β-Barrel Assembly Machine

“…Furthermore, a conformational cycle has been proposed for the BamA-assisted OMP folding and insertion that is modulated by mutation in some lipoprotein subunits of BAM (30). As BamA by itself has recently been reported to catalyze folding and insertion of substrate OMPs into liposomes in vitro (31), one important role of the BAM lipoproteins may be to modulate BamA conformation and increase the efficiency of the machinery (8). Therefore, understanding how the BAM lipoproteins interact with BamA is crucial to define their functional role.…”

“…High resolution structures of the individual BAM subunits have been reported (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Genetic and biochemical studies have shown that BamA interacts directly with BamB and BamD, whereas BamC and BamE are in contact with BamD (1,2,15,16).…”

The structure of the β-barrel assembly machinery complex

“…Subsequent studies showed that lateral opening of the barrel domain was required for function in BamA, strengthening an existing hypothesis that the barrel domain must open laterally in the membrane to allow insertion of the substrate OMPs into the OM (16, 18, 24). It has been proposed that BamB might serve as a scaffold, assisting in the handoff of nascent OMPs by SurA/Skp to BamA, while BamC, BamD, and BamE may serve support roles in regulating the function of BamA (14, 17, 25). The structures have offered clues to how each component may function within the complex; however, the lack of structural information regarding the fully assembled complex has hindered progress towards exploring the mechanism further.…”