LptB-LptF coupling mediates the closure of the substrate-binding cavity in the LptB2 FGC transporter through a rigid-body mechanism to extract LPS.
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ABSTRACT:
SUBMITTER: Lundstedt EA
PROVIDER: S-EPMC7529706 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The cell surface of the Gram-negative cell envelope contains lipopolysaccharide (LPS) molecules, which form a permeability barrier against hydrophobic antibiotics. The LPS transport (Lpt) machine composed of LptB2FGCADE forms a proteinaceous trans-envelope bridge that allows for the rapid and specific transport of newly synthesized LPS from the inner membrane (IM) to the outer membrane (OM). This transport is powered from the IM by the ATP-binding cassette transporter LptB2FGC. The ATP-driven cycling between closed- and open-dimer states of the ATPase LptB2 is coupled to the extraction of LPS by the transmembrane domains LptFG. However, the mechanism by which LPS moves from a substrate-binding cavity formed by LptFG at the IM to the first component of the periplasmic bridge, the periplasmic β-jellyroll domain of LptF, is poorly understood. To better understand how LptB2FGC functions in Escherichia coli, we searched for suppressors of a defective LptB variant. We found that defects in LptB2 can be suppressed by both structural modifications to the core oligosaccharide of LPS and changes in various regions of LptFG, including a periplasmic loop in LptF that connects the substrate-binding cavity in LptFG to the periplasmic β-jellyroll domain of LptF. These novel suppressors suggest that interactions between the core oligosaccharide of LPS and periplasmic regions in the transporter influence the rate of LPS extraction by LptB2FGC. Together, our genetic data reveal a path for the bi-directional coupling between LptB2 and LptFG that extends from the cytoplasm to the entrance to the periplasmic bridge of the transporter.IMPORTANCEGram-negative bacteria are intrinsically resistant to many antibiotics due to the presence of lipopolysaccharide (LPS) at their cell surface. LPS is transported from its site of synthesis at the inner membrane to the outer membrane by the Lpt machine. Lpt proteins form a transporter that spans the entire envelope and is thought to function similarly to a PEZ candy dispenser. This trans-envelope machine is powered by the cytoplasmic LptB ATPase through a poorly understood mechanism. Using genetic analyses in Escherichia coli, we found that LPS transport involves long-ranging bi-directional coupling across cellular compartments between cytoplasmic LptB and periplasmic regions of the Lpt transporter. This knowledge could be exploited in developing antimicrobials that overcome the permeability barrier imposed by LPS.
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Project description:In Gram-negative bacteria, lipopolysaccharide is essential for outer membrane formation and antibiotic resistance. The seven lipopolysaccharide transport (Lpt) proteins A-G move lipopolysaccharide from the inner to the outer membrane. The ATP-binding cassette transporter LptB2FG, which tightly associates with LptC, extracts lipopolysaccharide out of the inner membrane. The mechanism of the LptB2FG-LptC complex (LptB2FGC) and the role of LptC in lipopolysaccharide transport are poorly understood. Here we characterize the structures of LptB2FG and LptB2FGC in nucleotide-free and vanadate-trapped states, using single-particle cryo-electron microscopy. These structures resolve the bound lipopolysaccharide, reveal transporter-lipopolysaccharide interactions with side-chain details and uncover how the capture and extrusion of lipopolysaccharide are coupled to conformational rearrangements of LptB2FGC. LptC inserts its transmembrane helix between the two transmembrane domains of LptB2FG, which represents a previously unknown regulatory mechanism for ATP-binding cassette transporters. Our results suggest a role for LptC in achieving efficient lipopolysaccharide transport, by coordinating the action of LptB2FG in the inner membrane and Lpt protein interactions in the periplasm.
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Project description:Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE). The IM component LptB2FG powers the process through a yet unclarified mechanism. Here we report three high-resolution cryo-EM structures of LptB2FG alone and complexed with LptC (LptB2FGC), trapped in either the LPS- or AMP-PNP-bound state. The structures reveal conformational changes between these states and substrate binding with or without LptC. We identify two functional transmembrane arginine-containing loops interacting with the bound AMP-PNP and elucidate allosteric communications between the domains. AMP-PNP binding induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge. Functional assays reveal the functionality of the LptF and LptG periplasmic domains. Our findings shed light on the LPS transport mechanism.
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Project description:Lipopolysaccharide (LPS) is an essential glycolipid that covers the surface of gram-negative bacteria. The transport of LPS involves a dedicated seven-protein transporter system called the lipopolysaccharide transport system (Lpt) machinery that physically spans the entire cell envelope. The LptB2FG complex is an ABC transporter that hydrolyzes ATP to extract LPS from the inner membrane for transport to the outer membrane. Here, we extracted LptB2FG directly from the inner membrane with its original lipid environment using styrene-maleic acid polymers. We found that styrene-maleic acid polymers-LptB2FG in nanodiscs display not only ATPase activity but also a previously uncharacterized adenylate kinase (AK) activity, as it catalyzed phosphotransfer between two ADP molecules to generate ATP and AMP. The ATPase and AK activities of LptB2FG were both stimulated by the interaction on the periplasmic side with the periplasmic LPS transport proteins LptC and LptA and inhibited by the presence of the LptC transmembrane helix. We determined that the isolated ATPase module (LptB) had weak AK activity in the absence of transmembrane proteins LptF and LptG, and one mutation in LptB that weakens its affinity for ADP led to AK activity similar to that of fully assembled complex. Thus, we conclude that LptB2FG is capable of producing ATP from ADP, depending on the assembly of the Lpt bridge, and that this AK activity might be important to ensure efficient LPS transport in the fully assembled Lpt system.
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Project description:Secondary active transporters use electrochemical gradient of ions to fuel the "uphill" translocation of the substrate following the alternating-access model. The coupling of ions to conformational dynamics of the protein remains one of the least characterized aspects of the transporter function. We employ extended molecular dynamics (MD) simulations to examine the Na+-binding effects on the structure and dynamics of a LeuT-fold, Na+-coupled secondary transporter (Mhp1) in its major conformational states, i.e., the outward-facing (OF) and inward-facing (IF) states, as well as on the OF???IF state transition. Microsecond-long, unbiased MD simulations illustrate that Na+ stabilizes an OF conformation favorable for substrate association, by binding to a highly conserved site at the interface between the two helical bundles and restraining their relative position and motion. Furthermore, a special-protocol biased simulation for state transition suggests that Na+ binding hinders the OF???IF transition. These synergistic Na+-binding effects allosterically couple the ion and substrate binding sites and modify the kinetics of state transition, collectively increasing the lifetime of an OF conformation with high substrate affinity, thereby facilitating substrate recruitment from a low-concentration environment. Based on the similarity between our findings for Mhp1 and experimental reports on LeuT, we propose that this model may represent a general Na+-coupling mechanism among LeuT-fold transporters.
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Project description:Strong coupling between light and matter can occur when the interaction strength between a confined electromagnetic field and a molecular resonance exceeds the losses to the environment, leading to the formation of hybrid light-matter states known as polaritons. Ultrastrong coupling occurs when the coupling strength becomes comparable to the transition energy of the system. It is widely assumed that the confined electromagnetic fields necessary for strong coupling to organic molecules can only be achieved with external structures such as Fabry-Pérot resonators, plasmonic nanostructures, or dielectric resonators. Here we show experimentally that such structures are unnecessary and that a simple dielectric film of dye molecules supports sufficiently modified vacuum electromagnetic fields to enable room-temperature ultrastrong light-matter coupling. Our results may be of use in the design of experiments to probe polaritonic chemistry and suggest that polaritonic states are perhaps easier to realize than previously thought.
| S-EPMC8327311 | biostudies-literature
Project description:Background:Rigid plate fixation (RPF) is the cornerstone in managing fractures and osteotomies except for sternotomy, where most cardiac surgeons continue to use wire cerclage (WC). Results of a multicenter randomized trial evaluating sternal healing, sternal complications, patient reported outcome measures (PROMs), and costs after sternotomy closure with RPF or WC are summarized here. Methods:Twelve US centers randomized 236 patients to either RPF (n=116) or WC (n=120). The primary endpoint, sternal healing at 6 months, was evaluated by a core laboratory using computed tomography and a validated 6-point scale (greater scores represent greater healing). Secondary endpoints assessed through 6 months included sternal complications and PROMs. Costs from the time of sternal closure through 90 days and 6 months were analyzed by a health economic core laboratory. Results:RPF compared to WC resulted in better sternal healing scores at 3 (2.6±1.1 vs. 1.8±1.0; P<0.0001) and 6 months (3.8±1.0 vs. 3.3±1.1; P=0.0007) and higher sternal union rates at 3 [41% (42/103) vs. 16% (16/102); P<0.0001] and 6 months [80% (81/101) vs. 67% (67/100); P=0.03]. There were fewer sternal complications with RPF through 6 months [0% (0/116) vs. 5% (6/120); P=0.03] and a trend towards fewer sternal wound infections [0% (0/116) vs. 4.2% (5/120); P=0.06]. All PROMs including sternal pain, upper extremity function (UEF), and quality-of-life scores were numerically better in RPF patients compared to WC patients at all follow-up time points. Although RPF was associated with a trend toward higher index hospitalization costs, a trend towards lower follow-up costs resulted in total costs that were $1,888 less at 90 days in RPF patients compared to WC patients (95% CI: -$8,889 to $4,273; P=0.52) and $1,646 less at 6 months (95% CI: -$9,127 to $4,706; P=0.61). Conclusions:Sternotomy closure with RPF resulted in significantly better sternal healing, fewer sternal complications, improved PROMs and was cost neutral through 90 days and 6 months compared to WC.
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Project description:To characterize the conformational dynamics of sarcoplasmic reticulum (SR) calcium pump (SERCA) we performed molecular dynamics simulations beginning with several different high-resolution structures. We quantified differences in structural disorder and dynamics for an open conformation of SERCA versus closed structures and observed that dynamic motions of SERCA cytoplasmic domains decreased with decreasing domain-domain separation distance. The results are useful for interpretation of recent intramolecular Förster resonance energy transfer (FRET) distance measurements obtained for SERCA fused to fluorescent protein tags. Those previous physical measurements revealed several discrete structural substates and suggested open conformations of SERCA are more dynamic than compact conformations. The present simulations support this hypothesis and provide additional details of SERCA molecular mechanisms. Specifically, all-atoms simulations revealed large-scale translational and rotational motions of the SERCA N-domain relative to the A- and P-domains during the transition from an open to a closed headpiece conformation over the course of a 400 ns trajectory. The open-to-closed structural transition was accompanied by a disorder-to-order transition mediated by an initial interaction of an N-domain loop (N?5-?6, residues 426-436) with residues 133-139 of the A-domain. Mutation of three negatively charged N-domain loop residues abolished the disorder-to-order transition and prevented the initial domain-domain interaction and subsequent closure of the cytoplasmic headpiece. Coarse-grained molecular dynamics simulations were in harmony with all-atoms simulations and physical measurements and revealed a close communication between fluorescent protein tags and the domain to which they were fused. The data indicate that previous intramolecular FRET distance measurements report SERCA structure changes with high fidelity and suggest a structural mechanism that facilitates the closure of the SERCA cytoplasmic headpiece.
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