The TAM, a Translocation and Assembly Module for protein assembly and potential conduit for phospholipid transfer

Abstract

The assembly of β-barrel proteins into the bacterial outer membrane is an essential process enabling the colonization of new environmental niches. The TAM was discovered as a module of the β-barrel protein assembly machinery; it is a heterodimeric complex composed of an outer membrane protein (TamA) bound to an inner membrane protein (TamB). The TAM spans the periplasm, providing a scaffold through the peptidoglycan layer and catalyzing the translocation and assembly of β-barrel proteins into the outer membrane. Recently, studies on another membrane protein (YhdP) have suggested that TamB might play a role in phospholipid transport to the outer membrane. Here we review and re-evaluate the literature covering the experimental studies on the TAM over the past decade, to reconcile what appear to be conflicting claims on the function of the TAM.

Keywords: BAM Complex; Beta-barrel Proteins; Lipid Transport; Outer Membrane Biogenesis; TamB.

Figure 1. Domain architectures within the Omp85 and AsmA superfamilies of proteins.

(A) Diagrammatic representation of the domain structure of BamA, TamA, and the other eight Omp85 protein families found in bacteria (Heinz and Lithgow, 2014). N-terminal POTRA domains are shown in shades of green/blue, and the alternative N-terminal domains are shown in red, gray, or black as previously detailed (Heinz and Lithgow, 2014), and the β-barrel surface antigen domain is shaded with diagonal arrows. (B) The crystal structure of TamA (PDB: 4C00) (Gruss et al, 2013) and BamA (PDB: 5D0Q) (Noinaj et al, 2013) are shown portraying the arrangement of the POTRA domains with respect to the membrane-embedded β-barrel domain. (C) Diagrammatic representation of the domain structure of TamB and related AsmA-superfamily proteins are indicated, with the residue numbers shown at the C-terminus of each protein, the domains determined by CD-search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) are drawn to scale. (inset) The crystal structure of the β-taco fragment of TamB (PDB: 5VTG) (Josts et al, 2017), colored black, is shown diagrammatically in TamB as residues 975–1139. (D) Structure predictions for the E. coli representatives of the YhdP family, YdbH family, YhjG family, AsmA family YicH family and TamB family of proteins using Alphafold (Jumper et al, 2021). The predictions made use of default parameters and the color-coding is standard where dark blue represents the highest-confidence prediction and red-yellow denotes predictions of less confidence. In a similar analysis, the length projection of the periplasmic domain of YhdP was calculated to be 268 Å (Kumar and Ruiz, 2023). (Inset) The Alphafold prediction of the pseudosubstrate domain of TamB that mediates interaction with the N-terminal most POTRA domain of TamA, as classified by sequence features (Heinz et al, 2015) and biochemical analysis (Selkrig et al, ; Selkrig et al, 2012).

Figure 2. Models for the function of TamB.

(A) A model for TAM function drawing on measurements made by (Shen et al, 2014) and (Selkrig et al, 2015) to suggest dynamics in the movement of the TAM during its reaction cycle to assemble β-barrel proteins (red) after their translocation across the inner membrane (IM) is mediated by the SEC translocon. Measurement of the distance across the periplasm varies with an average that can be considered ~260 Å, with the peptidoglycan layer (PG) situated ~100 Å from the outer membrane (OM) (Asmar et al, ; Cohen et al, ; Mandela et al, 2022) and this scale is approximated in the diagram. The graphical representation of the topology of the TAM subunits, TamA and TamB, is based on (i) the extended TamB conformation (McDonnell et al, 2023) of 246 Å which would be necessary and sufficient to reach and contact TamA, and (ii) the crystal structure of TamA that has the POTRA domains approximately 44 Å from the outer membrane surface. Measurements by neutron reflectometry showed the POTRA domain is able to move to at least 77 Å from the membrane when it encounters its substrate (Shen et al, 2014). This movement of +33 Å would either locally deform the outer membrane (shown) or require a tilting of TamB to accommodate the downward movement. (B) A model for TAM function by analogy to the function of YhdP, where phospholipids have been cross-linked to the hydrophobic groove in YhdP (Cooper et al, 2023). By analogy, the hydrophobic groove in TamB might provide for equilibration of phospholipids between the inner membrane and outer membrane.