Lipid transport in Acinetobacter baumannii - Structure of the MlaBDEF complex

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Gram negative bacteria, like the critical hospital germ Acinetobacter baumannii, are enveloped by two lipid bilayers, shielding them effectively not only from mechanical forces, but also from a range of antibiotics like penicillin, detergents or enzymes like lysozyme. The two lipid bilayers are asymmetric, with the outside layer of the outer bilayer presenting lipopolysaccharides to the surface of the cell. The underlying molecular machinery that maintains lipid composition under consumption of considerable amounts of energy is therefore an attractive target for development of new antibiotic compounds. While the peripheral components of the MLA system were previously elucidated, the structural details of the core of the system, MlaBDEF, which is located in the inner membrane, remained previously elusive, although here the actual energy conversion from ATP into lateral transport presumably takes place.

We obtained cryo-EM maps for MlaBDEF bound to the ATP analogue AppNHP as well as ADP and the apo state in three different data collections at state-of-the-art 300 kV microscopes located at the Astbury Centre in Leeds and eBIC / Diamond Light Source after extensive condition-screening on our in-house 200 kV Tecnai Arctica Microscope "Ava the Arctica". Initial single particle analysis data processing in the software packages Relion and cisTEM was limited at the ab-initio 3D stage to about 5 Angstroms. Thanks to Mike Cianfrocco and Melanie Ohi, who organized a data processing workshop in Ann Arbor, Michigan we were introduced to the CryoSPARC software by Ali Punjani. The unique ab initio algorith together with non-uniform refinement managed to focus processing to the protein part and pushed resolution to about 4 Angstroms, which is a considerable improvement in CryoEM, as sidechains become visible at this resolution.

atomic structure of MlaBDEF
atomic structure of MlaBDEF

We built an atomic model of the 250 kDa protein complex and were fascinated by the complexity of the individual modules. At the cytosolic side MlaBDEF presents the paired ATPases MlaF, together with the subunits MlaB. Binding of MlaB occured in a flexible way, with 50% of the particles showing dual MlaB-MlaF binding, and the other 50% of the particles leaving one MlaB binding site unoccupied. The exact mechanism of this binding mode remains still elusive. AppNHP was bound at the peripheral interfaces of MlaF, MlaE and MlaD. The structure of paired MlaE presented a N-terminal helix that was parallel to the membrane instead of crossing it. At this position paired lipid binding sites are located, formed by the N-terminal and C-terminal helices of MlaE as well as the N-terminus of MlaD TM helix-1. At the cytosolic side of the MlaBDEF complex six MlaD copies form a hexagonal basket structure, with hydrophobic residues narrowing down a pore-like structure in the center. We also found densities for bound lipid residues in this region.

To learn more about the allocrit dynamics we performed a series of molecular dynamics simulations. When we removed bound lipids from the presumed cytosolic lipid binding pockets the spaces were rapidly re-occupied by bulk lipids, confirming them as lipid binding sites. Lipid dynamics in the periplasmic region were also insightful, as we could observe partial flipping of the centrally bound lipid moiety in the periplasmic pore. Taking together the CryoEM maps and the simulations we could gain an understanding of lipid dynamics in the MlaBDEF pore complex. You may find our full publication here: https://www.nature.com/articles/s42003-021-02318-4

Daniel Mann

Postdoc, Forschungszentrum Jülilch