Conformational Dynamics of the Lipopolysaccharide Transport Complex LptB2FGC Reveal Allosteric Regulation of Lipopolysaccharide Export

Core Concepts

The conformational heterogeneity and dynamic behavior of the lipopolysaccharide transport complex LptB2FGC, which is essential for the biogenesis of the outer membrane in Gram-negative bacteria, are allosterically regulated by ATP binding and hydrolysis to ensure efficient and unidirectional transport of lipopolysaccharide across the periplasm.

Abstract

The study investigates the conformational dynamics of the lipopolysaccharide (LPS) transport complex LptB2FGC using pulsed dipolar electron spin resonance (ESR) spectroscopy and laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). The key findings are:

The nucleotide-binding domains (NBDs) of LptB2FG exhibit coordinated conformational changes upon ATP binding and hydrolysis, closing the LPS binding pocket in the transmembrane domains (TMDs).

The lateral gate formed by LptG-TM1 and LptF-TM5, which is proposed to be the entry site for LPS, exhibits a highly dynamic and heterogeneous conformation even in the presence of nucleotides. Binding of LptC restricts the flexibility of this gate into two distinct conformations.

ATP binding allosterically opens the β-jellyroll domain of LptF, while the LptG β-jellyroll remains highly flexible, suggesting a selective regulation of these periplasmic domains.

The β-jellyroll domains of LptF and LptC form a stable interaction, potentially creating a continuous pathway for LPS transfer towards the outer membrane.

LILBID-MS data show that LPS is released from LptB2FG upon ATP hydrolysis, indicating that LptC is required for a productive transport cycle.

The results reveal the dynamic regulation of the LPS entry gate through the conformational changes of the LptC transmembrane helix, while its β-jellyroll domain is anchored in the periplasm. This, combined with the long-range ATP-dependent allosteric gating of the LptF β-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.

Stats

LptB2FG exhibits a mean interspin distance of ~5 nm between the nucleotide-binding domains in the apo state, which decreases to below 4 nm in the vanadate-trapped state.
The lateral gate formed by LptG-TM1 and LptF-TM5 exhibits a broad conformational distribution in the apo state, spanning the range of the open and closed structures.
The lateral gate formed by LptF-TM5 and LptG-TM1 shows a highly dynamic and heterogeneous conformation, even in the presence of nucleotides.

Quotes

"ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation."
"LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains."
"ATP binding allosterically opens the periplasmic LptF β-jellyroll domain, while the LptG β-jellyroll exhibits significant internal flexibility."

Key Insights Distilled From

Dynamic basis of lipopolysaccharide export by LptB2FGC

by Dajka,M., Ra... at www.biorxiv.org 05-21-2024

https://www.biorxiv.org/content/10.1101/2024.05.20.594984v2

Deeper Inquiries

How do the conformational dynamics of the LptB2FGC complex change in the presence of lipopolysaccharide substrates or inhibitors targeting the transport process

The conformational dynamics of the LptB2FGC complex change significantly in the presence of lipopolysaccharide substrates or inhibitors targeting the transport process. When LPS is present, the complex undergoes conformational changes that facilitate the binding, transfer, and release of LPS molecules. The interaction between the β-jellyroll domains of LptF and LptG stabilizes the complex and allows for the efficient transfer of LPS across the periplasm. Additionally, the lateral gates of the TMDs exhibit dynamic behavior, especially the second lateral gate, which is the putative entry site for LPS. This gate shows a large conformational heterogeneity, suggesting that it plays a crucial role in interacting with LPS and LptC. The presence of LptC further modulates the dynamics of the lateral gates, limiting their flexibility and creating an equilibrium between different conformations. Overall, the conformational dynamics of the LptB2FGC complex are finely tuned to ensure the efficient and unidirectional transport of LPS.

What are the potential counter-arguments to the proposed allosteric regulation mechanism, and how could alternative models be tested experimentally

Potential counter-arguments to the proposed allosteric regulation mechanism in the LptB2FGC complex could include alternative models that suggest different mechanisms for ATP binding and hydrolysis coupled with LPS transport. One counter-argument could be that the observed conformational changes are not directly related to ATP binding but rather to the presence of other factors in the experimental setup. To address this, control experiments could be conducted where ATP is omitted or replaced with non-hydrolysable analogs to confirm the role of ATP in inducing conformational changes. Another counter-argument could be that the observed dynamics are artifacts of the experimental conditions and do not accurately represent the native state of the complex. To address this, experiments could be performed in more physiologically relevant environments, such as native lipid bilayers, to validate the observed conformational dynamics. Alternative models could be tested experimentally by introducing mutations in key regions of the complex that are proposed to be involved in the allosteric regulation mechanism. By studying the effects of these mutations on the conformational dynamics and function of the complex, researchers can gain insights into the validity of the proposed mechanism and explore alternative models.

Given the importance of the lipopolysaccharide transport system in Gram-negative bacteria, how could the insights from this study be leveraged to develop novel antimicrobial strategies targeting this essential pathway

The insights from this study on the lipopolysaccharide transport system in Gram-negative bacteria could be leveraged to develop novel antimicrobial strategies targeting this essential pathway. One potential strategy could involve designing small molecules that specifically target the interactions between the β-jellyroll domains of LptF and LptG, disrupting the stability of the complex and inhibiting LPS transport. Another strategy could focus on developing inhibitors that interfere with the allosteric regulation mechanism of ATP binding and hydrolysis, thereby blocking the conformational changes necessary for LPS transfer. Additionally, the dynamic behavior of the lateral gates, especially the second lateral gate, could be targeted to prevent the entry of LPS into the complex. By understanding the detailed mechanisms of LPS transport and the conformational dynamics of the LptB2FGC complex, researchers can design targeted antimicrobial agents that disrupt this essential pathway in Gram-negative bacteria.