Structural basis of outer membrane protein insertion by the BAM complex

Abstract

3Outer membrane proteins (OMPs) play important roles in Gram-negative bacteria, mitochondria and chloroplasts in nutrition transport, protein import, secretion, and other fundamental biological processes [1][2][3] . Dysfunction of mitochondria outer membrane proteins are linked to disorders such as diabetes, Parkinsons and other neurodegenerative diseases 4,5 . The OMPs are inserted and folded correctly into the outer membrane (OM) by the conserved OMP85 family proteins [6][7][8] , suggesting that similar insertion mechanisms may be used in Gram-negative bacteria, mitochondria and chloroplasts.In Gram-negative bacteria, OMPs are synthesized in the cytoplasm, and are transported across the inner membrane by SecYEG into the periplasm 8,9 . The seventeen kilodalton (kDa) protein (Skp) and the survival factor A (SurA) chaperones escort the unfolded OMPs across the periplasm to the β-barrel assembly machinery (BAM), which is responsible for insertion and assembly of OMPs into the OM 10-12 . InEscherichia coli, the BAM complex consists of BamA and four lipoprotein subunits, BamB, BamC, BamD and BamE. BamA is comprised of five N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal OMP transmembrane barrel, while the four lipoproteins are affixed to the membrane by N-terminal lipid-modified cysteines. Of these subunits, BamA and BamD are essential 3,6 . One copy of each of these five proteins is required to form the BAM complex with an approximate molecular weight of 200 kDa (Extended Data Fig. 1). In vitro reconstitution of the E.coli BAM complex and functional assays showed that all five subunits are required to obtain the maximum activity of BAM [13][14][15][16] . Furthermore, comparison of the two complexes reveals that the periplasmic units are rotated with respect to the barrel, which appears to be linked to significant conformational changes in the β-strands β1C-β6C of the barrel. Taken together this suggests a novel insertion mechanism whereby rotation of the BAM periplasmic ring promotes insertion of OMPs into the OM. To our knowledge, this is the first reported crystal structure of an intramembrane barrel with a lateral-open conformation.Unique architecture of two E. coli BAM complexes X-ray diffraction data of selenomethionine labelled crystals were collected to 3.9Ångström (Å) resolution and the BAM structure was determined by singlewavelength anomalous dispersion (SAD) and manual molecular replacement (Methods, Extended Data Table 1). The first structure contained four proteins: BamA, BamC, BamD and BamE (Fig. 1a-c), with the electron density and crystal packing indicating that the BamB is absent in the complex. This was confirmed by SDS-PAGE analysis of the crystals (Extended Data Fig. 1 and Supplementary Data Fig. S1). In this model, BamA, BamC, BamD and BamE contain residues E22-I806, C25-K344, E26-S243, and C20-E110, respectively. The machinery is approximately 115 Å in length, 84 Å in width and 132 Å in height (Fig. 1a). 5The architecture of BamACDE resembles a top hat with a...

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

“…In terms of anaP2-anaP3, this is in line with earlier findings on BamA, where the C-terminal POTRA domains ecP3-ecP5 were assumed to have a rigid architecture (36). However, recent crystal structures of assembled BAM complexes challenge this assumption (8)(9)(10). In addition, the orientation of anaP2-anaP3 in the x-ray structure is well within this structure bundle, and thus seems to be a good representative of this ensemble.…”

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

“…Recent X-ray crystallography and cryo-EM studies reported the structures of several Omp85 family members, including FhaC, TamA, and BamA (28,(36)(37)(38)(43)(44)(45)(46)(47). Given the conservation of key features, these structures serve as models for Toc75, albeit with low sequence identity (7%, 7%, and 8%, respectively).…”

“…The structure reveals that the POTRA domains form an L-shaped structure very similar to BamA, with a unique extended helix within POTRA2 that is not observed in the POTRA domains of BamA and other Omp85 family members (25,28,(35)(36)(37)(38). Based on these unique structural properties, we investigated the contribution of individual POTRA repeats in binding chloroplast precursors.…”

“…BamA, one of the most well-characterized member of this family, has five POTRA domains residing in the periplasm, where they interact with nascent outer membrane protein substrates (23,26), additional components of the BAM complex (Bam B-E), and the periplasmic chaperone, SurA (27). It also has been suggested recently that the BamA POTRAs are involved in modulating the conformation of the BamA β-barrel to promote the insertion of nascent outer membrane proteins (28). In mitochondria, Sam50 is oriented with its single POTRA domain within the intermembrane space (IMS), where it interacts with substrates and promotes their release from Sam50 (29).…”

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

“…Although the structure of the Bam complex was recently solved by both X-ray crystallography and cryo-EM (13)(14)(15)(16), the mechanism by which it catalyzes the integration of b-barrel proteins into the OM remains poorly understood. The complex has the shape of a top hat in which the BamA bbarrel forms the crown and the POTRA domains together with the lipoproteins form a brim (14,15).…”