Investigation of the lipopolysaccharide transport system using electron spin resonance spectroscopy
Authors:   , Dajka Marina 
Published: December 2024

Biological membranes are essential for life, serving as barriers that separate the internal and external environments of cells. They regulate the passage of substances through selective permeability, establish ion gradients, and facilitate both energy production and cell communication. In bacterial cells, the membrane bilayer plays a crucial role in their antibiotic resistance.
Gram-negative bacteria possess a double bilayer as an inner membrane (IM) and an outer membrane (OM). The IM is composed of phospholipids (PL) and inner membrane proteins. The OM is an asymmetric bilayer composed of lipopolysaccharides (LPS) and PL with embedded outer membrane proteins (OMPs). The biogenesis of the OM is regulated by specialized machineries in the cell. The OMPs are transported, folded and inserted into the OM by the β-barrel assembly machinery (BAM) complex. The transport of LPS molecule and insertion into the OM is controlled by the lipopolysaccharide transport (Lpt) complex.
This dissertation focuses on the Lpt system, its function during hydrolysis and the interaction of the individual domains during LPS transport. The complex structure and function of this system is essential for OM formation. The LPS transport system consists of seven essential proteins (LptAnB2CDEFG) that span both the IM and OM. LPS is synthesized in individual components in the cytoplasm and subsequently transported to the periplasmic side of the IM by the proteins MsbA and Wzm/Wzt. In accordance with a PEZ candy dispenser model, the ATP binding cassette (ABC) transporter LptB2FG, situated within the IM, facilitates the transfer of LPS into the OM through the process of ATP hydrolysis. The LptB2FG complex laterally take up LPS, transfers it across the periplasmic β-jellyroll bridge, formed by LptA and LptC. Finally, LPS is pushed from LptA to the LptDE complex in the OM, where it is inserted into the OM. Recent insights into the mechanisms of LPS transport open up promising perspectives for the development of innovative therapies, especially for bacteria that are resistant to existing drugs. The ABC transporter LptB2FG is essential and consists mainly of the nucleotide binding domains (NBDs), the transmembrane domains (TMDs) and the β-jellyroll domains. An investigation of the above interactions was carried out, focusing on the influence of ATP binding and hydrolysis at the NBDs on substrate transport, TMD functionality and interdomain interactions of the β-jellyroll domains.
In this thesis, electron paramagnetic resonance (EPR) spectroscopy has been primarily used to study the Lpt complex. Pulsed EPR experiments such as Pulse Electron-Electron Double Resonance (PELDOR) are a successful tool for studying proteins by measuring protein distances between spin labels, covering a range of 1.5 to 16 nm. PELDOR is a valuable tool for studying membrane proteins in physiologically relevant contexts, requiring only micromolar concentrations of spin-labeled samples. It facilitates the understanding of protein conformations and provide information on the structural dynamics. Moreover the technique can be effectively combined with other biophysical techniques such as cryo-electron microscopy (cryo-EM), X-ray crystallography and NMR spectroscopy to provide global structural data.
The research included optimization of expression, membrane preparation, purification of LptB2FG, LptB2FG-C and LptDE complexes, as well as optimization of their spin-labeling and reconstitution into proteoliposomes (PLS). PELDOR spectroscopy was used to monitor conformational changes during LPS transport to better understand the function of LptB2FG and LptB2FG-C. Cryo-EM was used to provide insights into the LptB2FG structure at the hydrolysis state, while native mass spectrometry (MS) was used to monitor substrate binding and release. The results of these studies allow a comprehensive understanding of the process of LPS translocation and the interactions between the different protein components within the Lpt system.
The LptDE complex was expressed, purified and spin labeled successfully. The double mutant T236C-V747C was used to gain insights into the conformational states of the lateral gate in LptD, which is presumed to insert LPS into the OM. PELDOR measurements indicated that the gate of the protein is additionally open in the apo state. Further investigation of mutants could elucidate the interactions between the individual protein components of LptD, the loops, the LPS insertion, and LptE. Such studies could provide insights that would support the strategies to combat antibiotic-resistant bacteria and contribute to the development of new therapeutic approaches.