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Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical “corkscrew-like” membrane tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes. ESCRT-III complexes assemble in vivo inside membrane structures with a negative Gaussian curvature, but how membrane shape influences ESCRT-III polymerization remains unclear. Here authors use structural and biophysical methods to show how human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation.

Biosorption is an ingenious technique that uses biological materials to acquire trace metal ions from wastewater. In the present study, the ability of Colocasia esculenta stem biomass was explored for the biosorption of toxic trace metals. The maximum removal was observed for arsenate (As 5+ ) with 58.63%, followed by chromium (Cr 6+ ) with 56.56%, and cadmium (Cd 2+ ) with 41.2%. However, for copper (Cu 2+ ), nickel (Ni 2+ ), and zinc (Zn 2+ ), low adsorption was observed. Batch sorption tests revealed that adsorbent dosage of 0.5g, 0.5g, and 0.3g; time of 10 h, 4 h, and 10 h; room temperature range of 25–30°C; pH range of 7.0–4.5; and initial concentration of 30 μg/L, 20 mg/L, and 30 mg/L were the optimum conditions for the removal of As 5+ , Cr 6+ , and Cd 2+ , respectively. Scanning electron microscope and energy-dispersive X-ray spectroscopy (SEM-EDX) analysis of Colocasia esculenta stem biomass before and after adsorption revealed that the trace metals successfully get adsorbed on the surface of the biosorbent. The equilibrium data fitted well with the adsorption isotherm model of Langmuir (for As 5+ , Cr 6+ , and Cd 2+ ), Dubinin-Radushkevich (for As 5+ and Cr 6+ ), and Flory-Huggins (for Cd 2+ ), and the kinetic data of As 5+ , Cr 6+ , and Cd 2+ biosorption were best described by pseudo-second-order kinetic model. Thermodynamic studies revealed that the adsorption process for all concerned trace metals acts in a spontaneous manner and is endothermic in nature. Thus, the use of Colocasia esculenta stem biomass proved to be an efficient and economical alternative for the treatment of effluents contaminated with these trace metals.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance 1 – 3 . Teixobactin 4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan 5 . Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin 4 . The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates. Using a combination of methods, the mechanism of the antibiotic teixobactin is revealed.

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo-EM structures of membrane-coated CHMP2A–CHMP3 filaments from Homo sapiens of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A–CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence toward the tube interior. Inter-filament interactions are electrostatic, which may facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high-speed atomic force microscopy imaging corroborate that VPS4 can constrict and cleave CHMP2A–CHMP3 membrane tubes. We therefore conclude that CHMP2A–CHMP3–VPS4 act as a minimal membrane fission machinery. The cryo-EM structures of ESCRT-III CHMP2A and CHMP3 filaments reveal their mode of polymerization and interaction with negatively curved membrane. VPS4 constricts and cleaves the ESCRT-III CHMP2A–CHMP3 membrane tubes, thus acting as a minimal membrane fission machinery.

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscope (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment.

Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes. The mechanism of action of the antibacterial tripeptide AMC-109 is unclear. Here, Melcrová et al. show that AMC-109 self-assembles into stable aggregates with a cationic surface, and then individual peptides insert into the bacterial membrane and disrupt membrane nanodomains, thus affecting membrane function without forming pores.

Background ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. Results Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. Conclusions We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.

The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na⁺ ions. Static crystal structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of ∼0.5 nm, ∼1.0 nm, and ∼1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter GltPh. Since CitS and GltPh are structurally unrelated, we conclude that the three-state elevators have evolved independently.

This investigation characterized the relationship between percentage occurrences of three size classes of Hilsa juveniles (3–5, 5.1–10, and 10.1–15 cm) and six biogeochemical parameters (water temperature, chlorophyll-a, turbidity, salinity, dissolved oxygen, and pH) in the lower part of the tidal riverine to the entire estuarine stretch of Hooghly. These relationships helped us determine the ranges of each biogeochemical parameter for different suitability criteria in three seasons, pre-monsoon (February–May), monsoon (June–September), and post-monsoon (October–January). Salinity had the highest weight (0.37–0.39) followed by chlorophyll-a (0.26–0.30) and turbidity (0.12–0.20). The geospatial data of the biogeochemical parameters were interpolated followed by reclassification with the suitability ranges, separately for each season. These reclassified data were integrated into GIS-based modeling by a multi-criteria decision-making technique (analytical hierarchy process) to generate the habitat suitability maps of juvenile Hilsa. The model-derived information was verified with the indigenous knowledge of the fishers regarding the suitable habitat of juvenile Hilsa by conducting group discussions at 13 locations along the entire study area. The degree of agreement/disagreement between the model and the field information was determined by measuring Kendall’s tau (0.81–0.96) and Kappa coefficients (0.77–0.86), which indicated a strong agreement. In total, 3.80%, 10.12%, and 31.08% of the total river-estuarine area considered for the present study were identified as highly suitable for juvenile Hilsa during pre-monsoon, monsoon, and post-monsoon seasons, respectively. This mapping can act as baseline information for the policymakers for sustainable Hilsa fishing keeping in view the livelihood of fishers.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.