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Conformational Investigation of the Asymmetric Periplasmic Domains of E. coli LptB2FGC Using SDSL CW EPR Spectroscopy

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Abstract

The majority of pathogenic Gram-negative bacteria benefit from intrinsic antibiotic resistance, attributed primarily to the lipopolysaccharide (LPS) coating of the bacterial envelope. To effectively coat the bacterial cell envelope, LPS is transported from the inner membrane by the LPS transport (Lpt) system, which comprises seven distinct Lpt proteins, LptA–G, that form a stable protein bridge spanning the periplasm to connect the inner and outer membranes. The driving force of this process, LptB2FG, is an asymmetric ATP-binding cassette (ABC) transporter with a novel architecture and function that ejects LPS from the inner membrane and facilitates transfer to the periplasmic bridge. Here, we utilize site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy to probe conformational differences between the periplasmic domains of LptF and LptG. We show that LptC solely interacts with the edge β-strand of LptF and does not directly interact with LptG. We also quantify the interaction of periplasmic LptC with LptF. Additionally, we show that LPS cannot enter the protein complex externally, supporting the unidirectional LPS transport model. Furthermore, we present our findings that the presence of LPS within the LptB2FGC-binding cavity and the membrane reconstitution environment affect the structural orientation of the periplasmic domains of LptF and LptG, but overall are relatively fixed with respect to one another. This study provides insight into the structural asymmetry associated with the newly defined type VI ABC transporters class.

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Data availability

The data generated during the current study are included in this published article and its supplementary information file or available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank Kathryn Schultz for helpful discussions and training support and Karson Hilgendorf for general laboratory support. Research reported in this publication was supported by the National Institutes of Health (R01GM108817, S10OD011937, and S10OD025260).

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National Institutes of Health grants R01GM108817, S10OD011937, and S10OD025260.

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  1. Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA

    Nicholas P. Cina & Candice S. Klug

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Both authors contributed to the study conception and design, material preparation, and data analysis. Data were collected by N.C. The first draft of the manuscript was written by N.C. Both authors edited and approved the final manuscript.

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Correspondence to Candice S. Klug.

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Cina, N.P., Klug, C.S. Conformational Investigation of the Asymmetric Periplasmic Domains of E. coli LptB2FGC Using SDSL CW EPR Spectroscopy. Appl Magn Reson 55, 141–158 (2024). https://doi.org/10.1007/s00723-023-01590-3

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