Biology subjects

Junglas, B.

Publications and source records attributed to Junglas, B..

8 recordsLinked to original sources

bioRxiv2025

Membrane binding of a cyanobacterial ESCRT-III protein crucially involves the helix α1-3 hairpin conserved in all superfamily members

IM30, the inner membrane-associated protein of 30 kDa (also known as Vipp1) is essential for thylakoid membrane biogenesis and/or maintenance in chloroplasts and cyanobacteria. IM30 and its bacterial homolog PspA belong to the ESCRT-III superfamily, proteins previously thought to be restricted to eukaryotes and archaea. Despite low sequence similarity, IM30 shares key structural and functional features with eukaryotic ESCRT-IIIs, including a conserved 1-2 helical hairpin core and the ability to form oligomeric barrel- or rod assemblies that mediate membrane remodeling. Using IM30 variants, we now show that initial membrane recruitment of IM30 is driven by electrostatic interactions between the positively charged 1-3 helical hairpin and negatively charged lipid surfaces, paralleling the role of charged helical regions in some eukaryotic ESCRT-IIIs. This likely initiates lateral assembly of IM30 into higher-order barrel or rod structures on the membrane. Once assembled, 0 helices within these oligomers engage and stabilize internalized membrane tubules, mirroring membrane interaction strategies of eukaryotic ESCRT-IIIs, which use both N-terminal sequences and charged residues on 1/2. Thus, our findings demonstrate a conserved membrane binding and remodeling mechanism across the ESCRT-III superfamily, underscoring an evolutionary link in membrane dynamics between pro- and eukaryotes. SignificanceIM30, a membrane-associated protein found in cyanobacteria and chloroplasts, along with its bacterial homolog PspA, belongs to the ESCRT-III superfamily. Despite low sequence conservation, these proteins share structural and functional features with eukaryotic ESCRT-III proteins. We show that IM30 binds membranes via a conserved structural motif, followed by lateral assembly into higher-order complexes. This supports a mechanism of membrane remodeling that is conserved in prokaryotic and eukaryotic members of the ESCRT-III superfamily.

biochemistry↗

bioRxiv2025

The bacterial ESCRT-III PspA rods thin lipid tubules and and increase membrane curvature through helix α0 interactions

The phage shock protein A (PspA), a bacterial member of the ESCRT-III superfamily, forms rod-shaped helical assemblies that internalize membrane tubules. The N-terminal helix 0 of PspA (and other ESCRT-III members) has been suggested to act as a membrane anchor, the detailed mechanism, however, of how it binds to membranes and eventually triggers membrane fusion and/or fission events remains unclear. By solving a total of 15 cryo-electron microscopy (cryo-EM) structures of PspA and a truncation lacking the N-terminal helix 0 in the presence of EPL membranes, we show in molecular detail how PspA interacts with and remodels membranes: binding of the N-terminal helix 0 in the outer tubular membrane leaflet induces membrane curvature, supporting membrane tubulation by PspA. Detailed molecular dynamics simulations and free energy computations of interactions between the helix 0 and negatively charged membranes suggest a compensating mechanism between helix/membrane interactions and the energy contributions required for membrane bending. The energetic considerations are in line with the membrane structures observed in the cryo-EM images of tubulated membrane vesicles, fragmented vesicles inside tapered PspA rods, and shedded vesicles emerging at the thinner PspA rod ends. Our results provide insights into the molecular determinants and a potential mechanism of vesicular membrane remodeling mediated by a member of the ESCRT-III superfamily. SummaryThe rods of bacterial ESCRT-III protein PspA internalize and thin membrane tubules by overcoming the required bending energy through progressive membrane binding of the N-terminal helix in the PspA assembly.

biophysics↗

bioRxiv2025

CryoVIA - An image analysis toolkit for the quantification of membrane structures from cryo-EM micrographs

Imaging of lipid structures and associated protein complexes using electron cryo-microscopy (cryo-EM) is a common visualization and structure determination technique. The quantitative analysis of the membrane structures, however, is not routine and time consuming in particular when large amounts of data are involved. Here, we introduce the automated image-processing software cryo-vesicle image analyzer (CryoVIA) that parametrizes lipid structures of large data sets from cryo-EM images. This toolkit combines segmentation, structure identification with methods to automatically perform a large-scale data analysis of local and global membrane properties such as bilayer thickness, size, and curvature including membrane shape classifications. We included analyses of exemplary data sets of different lipid compositions and protein-induced lipid changes through an ESCRT-III membrane remodeling protein. The toolkit opens new possibilities to systematically study structural properties of membrane structures and their modifications from cryo-EM images.

bioinformatics↗

bioRxiv2024

Structural basis for Vipp1 membrane binding: From loose coats and carpetsto ring and rod assemblies

Vipp1 (also known as IM30) is essential in most oxygenic photoautotrophic organisms. It is involved in membrane remodeling and fusion and is critical for thylakoid membrane biogenesis and maintenance. Vipp1 has recently been identified as a member of the ESCRT-III superfamily of membrane remodeling proteins, albeit it still is elusive how Vipp1 interacts with and finally remodels membranes. Here we present a series of cryo-EM structures of cyanobacterial Vipp1 interacting with bacterial membranes: first, we solved seven structures between 5 and 7 [A] resolution of three unique helical and four types of stacked-ring assemblies engulfing membranes, and, second, using sub-tomogram averaging, we determined three [~]20 [A] resolution structures compatible with previously observed carpet structures at three different membrane curvatures. By analyzing ten additional unique structures of N-terminally truncated Vipp1, we could show that helix 0 is essential for membrane tubulation and forms the membrane anchoring domain of Vipp1. Using a conformation-restrained Vipp1 mutant, we were able to reduce the structural plasticity of Vipp1 assemblies in the presence of lipids and determined two structures of Vipp1 at 3.0 [A] resolution, resolving the molecular details of membrane anchoring and intersubunit contacts of helix 0. Our data reveal the molecular details of how Vipp1 interacts with membranes, showing membrane curvature-dependent structural transitions from carpets to rings and rods, some of which are capable of inducing and/or stabilizing high local membrane curvature triggering membrane fusion. SummaryBacterial ESCRT-III family member Vipp1 forms membrane-bound coats, carpets, ring complexes, stacked-ring assemblies and helical tubes capable of internalizing lipids and inducing high membrane curvature.

biophysics↗

bioRxiv2024

Structural plasticity of bacterial ESCRT-III protein PspA in higher-orderassemblies

Eukaryotic members of the endosome sorting complex required for transport III (ESCRT-III) family have been shown to form diverse oligomeric assemblies. The bacterial phage shock protein A (PspA) has recently been identified as a bacterial member of the ESCRT-III superfamily, and monomeric PspA homo-oligomerizes to form large rod-shaped assemblies. As observed for eukaryotic ESCRT-III, PspA forms different tubular assemblies with varying diameters. Using electron cryo-microscopy (cryo-EM), we determined a total of 61 PspA structures and observed in molecular detail how structural plasticity of PspA rods is mediated by conformational changes at three hinge regions in the monomer and by the fixed as well as changing molecular contacts between protomers. Moreover, we reduced and increased the structural plasticity of PspA rods by removing the loop connecting helices 3/4 and the addition of nucleotides, respectively. Based on our analysis of PspA-mediated membrane remodeling, we suggest that the observed mode of structural plasticity is a prerequisite for the biological function of ESCRT-III superfamily members. SummaryA series of cryo-EM structures of PspA rods with induced diameter modulations reveals the molecular basis of structural plasticity.

biophysics↗

bioRxiv2020

Mg2+-binding shifts the IM30 activity from membrane protection to membrane destabilization

The inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. A soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting processes have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. The latter process depends on Mg2+-binding to IM30; yet, it still is unclear how Mg2+-loaded IM30 interacts with membranes and promotes membrane fusion. Here we show that interaction of Mg2+ with IM30 results in increased binding of IM30 to native as well as model membranes. Via Atomic Force Microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in presence of Mg2+. The observed interaction of IM30 with membrane surfaces differs dramatically from previously observed membrane-stabilizing, carpet-like structures in the absence of Mg2+. Mg2+-induced alterations of the IM30 structure switches the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.

biochemistry↗

bioRxiv2020

PspA maintains membrane integrity by an ESCRT-like membrane remodeling activity

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 [A] resolution cryo-EM structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generate positive membrane curvature. Using cryo-EM we visualized how PspA remodels membrane vesicles into m-sized structures and how it mediates the formation of internalized vesicular structures. Hot spots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

microbiology↗

bioRxiv2020

IM30 IDPs form a membrane protective carpet upon super-complex disassembly

Members of the phage shock protein A (PspA) family, including the inner membrane-associated protein of 30 kDa (IM30), are suggested to stabilize stressed cellular membranes. Furthermore, IM30 is essential in thylakoid membrane-containing chloroplasts and cyanobacteria, where it is involved in membrane biogenesis and/or remodeling. While it is well known that PspA and IM30 bind to membranes, the mechanism of membrane stabilization is still enigmatic. Here we report that ring-shaped IM30 super-complexes disassemble on membranes, resulting in formation of a membrane-protecting protein carpet. Upon ring dissociation, the C-terminal domain of IM30 unfolds, and the protomers self-assemble on membranes. IM30 assemblies at membranes have been observed before in vivo and were associated to stress response in cyanobacteria and chloroplasts. These assemblies likely correspond to the here identified carpet structures. Our study defines the thus far enigmatic structural basis for the physiological function of IM30 and related proteins, including PspA, and highlights a hitherto unrecognized concept of membrane stabilization by intrinsically disordered proteins.

biophysics↗