Rath, T. | Search bioRxiv

bioRxiv2024

Dynamic basis of lipopolysaccharide export by LptB2FGC

Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The {beta}-jellyroll domain of LptF stably interacts with the LptG and LptC {beta}-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF {beta}-jellyroll domain. In LptB2FG, 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 en try 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. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its {beta}-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF {beta}-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.

biophysics↗

bioRxiv2023

Lateral gating mechanism and plasticity of the BAM complex in micelles and E. coli

The {beta}-barrel assembly machinery (BAM) mediates folding and insertion of the majority of OMPs in Gram-negative bacteria. BAM is a penta-heterooligomeric complex consisting of the central {beta}-barrel BamA and four interacting lipoproteins BamB, C, D, and E. The conformational switching of BamA between inward-open (IO) and lateral-open (LO) conformations is required for substrate recognition and folding. However, the mechanism for the lateral gating or how the structural details observed in vitro correspond with the cellular environment remains elusive. Here we addressed these questions by characterizing the conformational heterogeneity of BamAB, BamACDE and BamABCDE complexes in detergent micelles and or E. coli using pulsed dipolar electron spin resonance spectroscopy (PDS). We show that the binding of BamB does not induce any visible changes in BamA and the BamAB complex exists in the IO conformation. The BamCDE complex induces an IO to LO transition through a coordinated movement along the BamA barrel. However, the extracellular loop (L6) is unaffected by the presence of lipoproteins and exhibits a large segmental dynamics extending to the exit pore. PDS experiments with BamABCDE complex in intact E. coli confirmed the dynamic behavior of both the lateral gate and the L6 in the native environment. Our results demonstrate that the BamCDE complex plays a key role for the function by regulating lateral gating in BamA.

biophysics↗