Oral Presentation 51st Lorne Proteins Conference 2026

The Basis of Phospholipid Transport to the Bacterial Outer Membrane by the Translocation and Assembly Module (132632)

Lachlan S R Adamson 1 , Alanah G Eisenhuth 1 , Ghaeath S K Abbas 1 , Rachel A North 1 , Denisse L Leyton 2 , Constance B Bailey 1 , Alastair G Stewart 3 , Catherine Zhang 1 , Anthony S Don 1 , Harris D Bernstein 4 , Aidan B Grosas 5 , Matthew T Doyle 1
  1. The University of Sydney, Camperdown, NSW, Australia
  2. Australian National University, Canberra, ACT, Australia
  3. Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia
  4. National Institutes of Health, Bethesda, MD, USA
  5. University of Wollongong, Wollongong, NSW, Australia

The outer membrane of gram-negative bacteria is composed of phospholipids, proteins, and lipopolysaccharide. The balance of all these components is essential for outer membrane integrity and cell viability. The TAM complex has been reported to be involved both in the insertion of proteins into the outer membrane1,2 and alternately as a transporter of phospholipids from the inner membrane to the outer membrane.3 The complex consists of TamA, an outer-membrane embedded β-barrel; and TamB, a periplasm-spanning protein possessing a hydrophobic β-taco channel, and a C-terminal domain of unknown function. Knocking out of the TAM complex has been reported to disrupt the insertion of outer-membrane proteins.1 The knocking out of TamB, in combination with related ‘AsmA-like’ proteins, has been shown to disrupt phospholipid transport to the outer membrane.3 Structures of the individual components of the TAM complex have been reported,4,5 but it is unknown how the two come into complex.

This work reports the cryoEM structure of TamA in complex with a truncated TamB bearing the domain of unknown function. We show that the C-terminus of TamB forms an unprecedented stable ‘hybrid-barrel’ with TamA by forming beta-sheets with the C-terminal strands of the TamA barrel. We use 3D variability analysis to identify a dynamic loop in TamB and use a combination of disulfide-crosslinking and phenotypic assay to show that the dynamicity of the loop is integral to outer membrane integrity. Combined these results provide insight into the function of the TAM complex and highlight its importance in the maintenance of the outer membrane.

  1. (1) Discovery of an archetypal protein transport system in bacterial outer membranes | Nature Structural & Molecular Biology. https://www.nature.com/articles/nsmb.2261#citeas (accessed 2025-08-28).
  2. (2) Wang, X.; Nyenhuis, S. B.; Bernstein, H. D. The Translocation Assembly Module (TAM) Catalyzes the Assembly of Bacterial Outer Membrane Proteins in Vitro. Nat Commun 2024, 15 (1), 7246. https://doi.org/10.1038/s41467-024-51628-8.
  3. (3) Absence of YhdP, TamB, and YdbH leads to defects in glycerophospholipid transport and cell morphology in Gram-negative bacteria | PLOS Genetics.
  4. (4) Josts, I.; Stubenrauch, C. J.; Vadlamani, G.; Mosbahi, K.; Walker, D.; Lithgow, T.; Grinter, R. The Structure of a Conserved Domain of TamB Reveals a Hydrophobic β Taco Fold. Structure 2017, 25 (12), 1898-1906.e5. https://doi.org/10.1016/j.str.2017.10.002.
  5. (5) Gruss, F.; Zähringer, F.; Jakob, R. P.; Burmann, B. M.; Hiller, S.; Maier, T. The Structural Basis of Autotransporter Translocation by TamA. Nat Struct Mol Biol 2013, 20 (11), 1318–1320. https://doi.org/10.1038/nsmb.2689.