Biology subjects

Hellmann, N.

Publications and source records attributed to Hellmann, N..

7 recordsLinked to original sources

bioRxiv2026

RNA G-quadruplexes mediate cooperativity in HNRNPH binding and splicing regulation

Alternative splicing, regulated by RNA-binding proteins (RBPs), enables the generation of diverse transcript isoforms critical for cellular function. However, how RNA secondary structure impacts RBP binding and function remains poorly understood. Here, we unravel how RNA G-quadruplexes (rG4s) facilitate cooperativity in splicing regulation by the RBP heterogeneous nuclear ribonucleoprotein H (HNRNPH). Through high-throughput in vivo and in vitro studies combined with theoretical modeling, we dissect how rG4s mediate cooperative HNRNPH binding to RNA, ultimately modulating the splicing of hundreds of exons. rG4 unfolding by HNRNPH exposes multiple G-rich binding sites, thereby establishing indirect cooperativity, which is further amplified to achieve switch-like splicing regulation. HNRNPH-mediated regulation is evident in breast cancer patients, with tumors showing rG4-disrupting variants and global HNRNPH alterations, driving distinct splicing patterns that distinguish tumor subtypes. Overall, our findings offer valuable insights into the mechanistic role of RNA secondary structures in cooperative RBP binding and splicing regulation and highlight the clinical relevance of HNRNPH-dependent splicing in cancer. HighlightsO_LIHundreds of cassette exons are cooperatively regulated by HNRNPH. C_LIO_LIUnfolding of RNA G-quadruplexes (rG4s) at HNRNPH binding sites facilitates indirect cooperativity in RNA binding. C_LIO_LIMulti-step splicing amplifies the response into highly switch-like regulation. C_LIO_LIrG4-disrupting variants and changing HNRNPH expression are associated with breast cancer phenotypes. C_LI

molecular biology↗

bioRxiv2026

The structured hairpin region of the bacterial ESCRT-III protein IM30 orchestrates stress-induced condensate formation

Biomolecular condensates are well characterized in eukaryotes, but their role in bacteria remains largely elusive. In the cyanobacteriumn Synechocystis sp. PCC 6803, the protein IM30, a member of the ESCRT-III superfamily of membrane remodeling proteins, forms stress-induced puncta across diverse environmental challenges, indicating a general adaptive response. Live-cell imaging reveals that IM30 is uniformly distributed throughout the cytoplasm at low concentrations but assembles into puncta upon exceeding a critical saturation threshold, a hallmark of liquid-liquid phase separation. Crucially, stress triggers puncta formation even below this threshold, suggesting stress lowers the phase-separation barrier. Super-resolution microscopy confirms spherical, condensate-like morphologies, while FRAP demonstrates rapid fluorescence recovery, consistent with with a fluid interior and dynamic exchange between puncta and the cytosol. Cellular IM30 levels exceed the in vitro determined critical concentration, placing the protein in a supersaturated state primed for condensation. Domain mapping identifies the structured 1-3 helical hairpin as the minimal phase separation driver; in contrast, the disordered 4-6 segment alone cannot phase-separate. Phase separation occurs within a physiologically relevant pH range (4.5-6.5), matching the localized acidification of the cyanobacterial cytoplasm at damaged thylakoid membranes. This directly links membrane stress, pH changes, and IM30 recruitment. Collectively, these findings establish IM30 puncta as bona fide, stress-responsive biomolecular condensates that function as rapid stress sensors and effectors, providing a mechanistic framework for phase separation and condensate formation by bacterial ESCRT-III proteins during environmental adaptation.

biochemistry↗

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↗

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↗

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

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↗