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.

Drug uptake is constrained by epithelial barriers, such as the intestine and the blood-brain barrier, which express efflux pumps such as the ABC-transporter ABCB1. Therefore, drugs targeting the central nervous system frequently require co-administration of efflux inhibitors to facilitate sufficient target engagement. Conversely, certain drugs designed to target distinct molecular pathways may inadvertently affect ABC transporters. However, detailed functional studies of putative human ABCB1 inhibitors is challenging due to difficulties in obtaining sufficiently high amounts of protein needed. Bacterial ABC transporters, such as BmrA from Bacillus subtilis, share structural and functional similarities with ABCB1 and thus provide a reasonable alternative model system. This study investigates clomipramine, an antidepressant known to reduce multidrug resistance in human cells by a yet undefined mechanism. We demonstrate that clomipramine inhibits the ATPase activity of both full-length BmrA and its isolated nucleotide-binding domain. Using binding assays and molecular modeling, multiple clomipramine binding sites were identified in BmrA, which are also conserved in human ABCB1. Critically, one predicted site overlaps with the ATP-binding pocket. The results underscore the potential side-effects of clomipramine and highlight the value of bacterial models such as BmrA for studying novel inhibitors when studies on human proteins are not feasible.

IM30, the inner membrane-associated protein of 30 kDa, conserved in cyanobacteria and chloroplasts, is a member of the ESCRT-III superfamily of membrane remodeling proteins. Like other ESCRT-III proteins, IM30 forms higher-order oligomeric structures, although the mechanisms regulating its assembly and disassembly remain poorly understood. A hallmark of ESCRT-III protein monomers is the presence of at least five α-helices, with the long helices α1 and α2/3 forming a helical hairpin that constitutes the structural core of all superfamily members. In contrast to eukaryotic ESCRT-III subunits, helices α0 and α4-α6 of Synechocystis IM30 unfold upon oligomer disassembly. Given that intrinsically disordered proteins often form biomolecular condensates via liquid-liquid phase separation and IM30 has previously been observed to form puncta structures in vivo under membrane stress, we here investigated whether IM30 has the ability to form biomolecular condensates in vitro. We demonstrate that IM30 forms condensates under physiologically relevant conditions of salt and protein concentrations, suggesting a functional link between the now observed condensate formation and membrane dynamics. Condensate formation is driven by the polyampholyte nature of IM30, yielding condensates that can be dissolved by both high and low salt concentrations. In living cyanobacterial cells, we observed puncta structures under salt stress, which we now link to the formation of condensates. We propose that condensates serve as transient hubs, locally concentrating IM30 monomers under stress conditions without requiring energy-intensive disassembly of preformed oligomers. Thus, condensate formation may represent a crucial early step in IM30-mediated stress response in bacteria and chloroplasts.

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 α 1– α 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 membrane binding of IM30 is driven by electrostatic interactions between the positively charged α1–α3 helical hairpin and negatively charged lipid surfaces, paralleling the role of charged helical regions in some eukaryotic ESCRT‐IIIs. This likely is followed by lateral assembly of IM30 into higher‐order barrel or rod structures on the membrane. Once assembled, α0 helices within these oligomers engage and stabilize internalized membrane tubules, mirroring membrane interaction strategies of eukaryotic ESCRT‐IIIs, which use both N‐terminal sequence motifs and charged residues on α 1/ α 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.

The phage shock protein A (PspA), a bacterial member of the endosomal sorting complexes required for transport (ESCRT)-III superfamily, forms rod-shaped helical assemblies that internalize membrane tubules. The N-terminal helix α0 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 cryoelectron microscopy (cryo-EM) structures of PspA and a truncation lacking the N-terminal helix α0 in the presence of Escherichia coli polar lipid membranes, we show in molecular detail how PspA interacts with and remodels membranes: Binding of the N-terminal helix α0 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 α0 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.

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 Å resolution of three unique helical and four types of stacked-ring assemblies engulfing membranes, and, second, using sub-tomogram averaging, we determined three ∼20 Å 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 α0 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 Å resolution, resolving the molecular details of membrane anchoring and intersubunit contacts of helix α0. 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. Summary Bacterial 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. ### Competing Interest Statement The authors have declared no competing interest.

SynDLP, a dynamin-like protein (DLP) encoded in the cyanobacterium Synechocystis sp. PCC 6803, has recently been identified to be structurally highly similar to eukaryotic dynamins. To elucidate structural changes during guanosine triphosphate (GTP) hydrolysis, we solved the cryoelectron microscopy (cryo-EM) structures of oligomeric full-length SynDLP after addition of guanosine diphosphate (GDP) at 4.1 Å and GTP at 3.6-Å resolution as well as a GMPPNP-bound dimer structure of a minimal G-domain construct of SynDLP at 3.8-Å resolution. In comparison with what has been seen in the previously resolved apo structure, we found that the G-domain is tilted upward relative to the stalk upon GTP hydrolysis and that the G-domain dimerizes via an additional extended dimerization domain not present in canonical G-domains. When incubated with lipid vesicles, we observed formation of irregular tubular SynDLP assemblies that interact with negatively charged lipids. Here, we provide the structural framework of a series of different functional SynDLP assembly states during GTP turnover.

The inner membrane associated protein of 30 kDa (IM30), a member of the endosomal sorting complex required for transport (ESCRT-III) superfamily, is crucially involved in the biogenesis and maintenance of thylakoid membranes in cyanobacteria and chloroplasts. In solution, IM30 assembles into various large oligomeric barrel- or tube-like structures, whereas upon membrane binding it forms large, flat carpet structures. Dynamic localization of the protein in solution, to membranes and changes of the oligomeric states are crucial for its in vivo function. ESCRT-III proteins are known to form oligomeric structures that are dynamically assembled from monomeric/smaller oligomeric proteins, and thus these smaller building blocks must be assembled sequentially in a highly orchestrated manner, a still poorly understood process. The impact of IM30 oligomerization on function remains difficult to study due to its high intrinsic tendency to homo-oligomerize. Here, we used molecular dynamics simulations to investigate the stability of individual helices in IM30 and identified unstable regions that may provide structural flexibility. Urea-mediated disassembly of the IM30 barrel structures was spectroscopically monitored, as well as changes in the protein's tertiary and secondary structure. The experimental data were finally compared to a three-state model that describes oligomer disassembly and monomer unfolding. In this study, we identified a highly stable conserved structural core of ESCRT-III proteins and discuss the advantages of having flexible intermediate structures and their putative relevance for ESCRT-III proteins.

Vesicle-inducing protein in plastids 1 (Vipp1) is critical for thylakoid membrane biogenesis and maintenance. Although Vipp1 has recently been identified as a member of the endosomal sorting complexes required for transport III superfamily, it is still unknown how Vipp1 remodels membranes. Here, we present cryo-electron microscopy structures of Synechocystis Vipp1 interacting with membranes: seven structures of helical and stacked-ring assemblies at 5-7-Å resolution engulfing membranes and three carpet structures covering lipid vesicles at ~20-Å resolution using subtomogram averaging. By analyzing ten structures of N-terminally truncated Vipp1, we show that helix α0 is essential for membrane tubulation and forms the membrane-anchoring domain of Vipp1. Lastly, using a conformation-restrained Vipp1 mutant, we reduced the structural plasticity of Vipp1 and determined two structures of Vipp1 at 3.0-Å resolution, resolving the molecular details of membrane-anchoring and intersubunit contacts of helix α0. Our data reveal 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.

Cardiolipin, the mitochondria marker lipid, is crucially involved in stabilizing the inner mitochondrial membrane and is vital for the activity of mitochondrial proteins and protein complexes. Directly targeting cardiolipin by a chemical-biology approach and thereby altering the cellular concentration of "available" cardiolipin eventually allows to systematically study the dependence of cellular processes on cardiolipin availability. In the present study, physics-based coarse-grained free energy calculations allowed us to identify the physical and chemical properties indicative of cardiolipin selectivity and to apply these to screen a compound database for putative cardiolipin-binders. The membrane binding properties of the 22 most promising molecules identified in the in silico approach were screened in vitro, using model membrane systems finally resulting in the identification of a single molecule, CLiB (CardioLipin-Binder). CLiB clearly affects respiration of cardiolipin-containing intact bacterial cells as well as of isolated mitochondria. Thus, the structure and function of mitochondrial membranes and membrane proteins might be (indirectly) targeted and controlled by CLiB for basic research and, potentially, also for therapeutic purposes.

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. The 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 functions have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. IM30's fusogenic activity 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 the 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 the presence of Mg2+. These structures differ dramatically from the membrane-stabilizing carpet structures that were previously observed in the absence of Mg2+. Thus, Mg2+-induced alterations of the IM30 structure switch the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.

Genetic assays capable of measuring the propensity of transmembrane helices to oligomerize within the cytoplasmic membrane of the bacterium E. coli are frequently used when sequence-specificity in transmembrane helix-helix interactions is investigated. In the present study, dimerization of the well-investigated wild-type and G83I-mutated transmembrane helix of the human glycophorin A protein was studied. Gradual prolongation of the transmembrane helix at the C-terminus with Leu residues lead to pronounced changes in the dimerization propensity when measured with the TOXCAT assay. Thus, besides sequence specificity, hydrophobic mismatch between the hydrophobic core of a studied transmembrane helix and the E. coli membrane can impact the oligomerization propensity of a transmembrane helix. This suggests that the results of genetic assays aiming at determining interactions of heterologous transmembrane helices within the E. coli membrane do not necessarily solely reflect sequence specificity in transmembrane helix-helix interactions, but might be additionally modulated by topological and structural effects caused by hydrophobic mismatch.

Complex formation of membrane-bound sensor kinases with the response regulators represents an inherent step of signaling from the membrane to the promoters on the DNA. In the C4-dicarboxylate-sensing DcuS-DcuR two-component system, complex formation is strengthened by activation (phosphorylation) in vitro and in vivo, with trapping of the response regulator DcuR at the membrane. ABSTRACT Signaling of two-component systems by phosphoryl transfer requires interaction of the sensor kinase with the response regulator. Interaction of the C4-dicarboxylate-responsive and membrane-integral sensor kinase DcuS with the response regulator DcuR was studied. In vitro, the cytoplasmic part of DcuS (PASC-Kin) was employed. Stable complexes were formed, when either DcuS or DcuR were phosphorylated (Kd 22 ± 11 and 28 ± 7 nM, respectively). The unphosphorylated proteins produced a more labile complex (Kd 1380 ± 395 nM). Bacterial two-hybrid studies confirm interaction of DcuR with DcuS (and PASC-Kin) in vivo. The absolute contents of DcuR (197-979 pmol mg−1 protein) in the bacteria exceeded those of DcuS by more than 1 order of magnitude. According to the Kd values, DcuS exists in complex, with phosphorylated but also unphosphorylated DcuR. In live cell imaging, the predominantly freely diffusing DcuR becomes markedly less mobile after phosphorylation and activation of DcuS by fumarate. Portions of the low mobility fraction accumulated at the cell poles, the preferred location of DcuS, and other portions within the cell, representing phosphorylated DcuR bound to promoters. In the model, acitvation of DcuS increases the affinity toward DcuR, leading to DcuS-P × DcuR formation and phosphorylation of DcuR. The complex is stable enough for phosphate-transfer, but labile enough to allow exchange between DcuR from the cytosol and DcuR-P of the complex. Released DcuR-P diffuses to target promoters and binds. Uncomplexed DcuR-P in the cytosol binds to nonactivated DcuS and becomes dephosphorylated. The lower affinity between DcuR and DcuS avoids blocking of DcuS and allows rapid exchange of DcuR. IMPORTANCE Complex formation of membrane-bound sensor kinases with the response regulators represents an inherent step of signaling from the membrane to the promoters on the DNA. In the C4-dicarboxylate-sensing DcuS-DcuR two-component system, complex formation is strengthened by activation (phosphorylation) in vitro and in vivo, with trapping of the response regulator DcuR at the membrane. Single-molecule tracking of DcuR in the bacterial cell demonstrates two populations of DcuR with decreased mobility in the bacteria after activation: one at the membrane, but a second in the cytosol, likely representing DNA-bound DcuR. The data suggest a model with binding of DcuR to DcuS-P for phosphorylation, and of DcuR-P to DcuS for dephosphorylation, allowing rapid adaptation of the DcuR phosphorylation state. DcuR-P is released and transferred to DNA by 3D diffusion.

Human Claudin-7 (Cldn7) is a member of the Claudin (Cldn) superfamily. In vivo, these proteins form tight junctions, which establish constricted connections between cells. Cldns oligomerize within the membrane plane (= cis-interaction), and also interact with Cldns from adjacent cells (= trans-interaction). Interactions of Cldns are typically studied in vivo and structural analyses of isolated Cldns are limited. Here, we describe heterologous expression in E. coli and purification of human Cldn7, enabling in vitro analyses of the isolated protein using detergent and model membrane systems. Cldn7 exists as a monomer, hexamer, and various higher oligomers in micelles. While only limited unfolding of the protein was observed in the presence of the anionic detergent sodium dodecyl sulfate, decreased ionic strength did affect Cldn7 cis-interactions. Furthermore, we identified two amino acids which mediate electrostatic cis-interactions and analyzed the impact of disturbed cis-interaction on trans-contacts via atomic force microscopy and monitoring Förster resonance energy transfer between fluorescently labeled Cldn7-containing proteoliposomes. Our results indicate that Cldn7 cis-oligomerization might not be a prerequisite for establishing trans-contacts.

IM30, the inner membrane‐associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo‐oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.

Summary 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 Å 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 μm-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.

The hydrophobic tails of aliphatic primary alcohols do insert into the hydrophobic core of a lipid bilayer. Thereby, they disrupt hydrophobic interactions between the lipid molecules, resulting in a decreased lipid order, i.e., an increased membrane fluidity. While aromatic alcohols, such as 2-phenylethanol, also insert into lipid bilayers and disturb the membrane organization, the impact of aromatic alcohols on the structure of biological membranes, as well as the potential physiological implication of membrane incorporation has only been studied to a limited extent. Although diverse targets are discussed to be causing the bacteriostatic and bactericidal activity of 2-phenylethanol, it is clear that 2-phenylethanol severely affects the structure of biomembranes, which has been linked to its bacteriostatic activity. Yet, in fungi some 2-phenylethanol derivatives are also produced, some of which appear to also have bacteriostatic activities. We showed that the 2-phenylethanol derivatives phenylacetic acid, phenyllactic acid, and methyl phenylacetate, but not Tyrosol, were fully incorporated into model membranes and affected the membrane organization. Furthermore, we observed that the propensity of the herein-analyzed molecules to partition into biomembranes positively correlated with their respective bacteriostatic activity, which clearly linked the bacteriotoxic activity of the substances to biomembranes.

The thermodynamic and kinetic principles guiding folding of transmembrane (TM) proteins are still not very well understood, at least when compared with soluble proteins. However, there is a considerable interest in better understanding these processes, not just since membrane proteins are important drug targets. Membrane proteins constitute ∼25–30% of total proteins encoded in any organism, and many diseases are caused by alterations in TM protein structure and function ( 1 Wallin E. von Heijne G. Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 1998; 7: 1029-1038 Crossref PubMed Scopus (1229) Google Scholar , 2 Marinko J.T. Huang H. Sanders C.R. et al. Folding and misfolding of human membrane proteins in health and disease: from single molecules to cellular proteostasis. Chem. Rev. 2019; 119: 5537-5606 Crossref PubMed Scopus (97) Google Scholar ). The thermodynamic stability of soluble proteins is classically studied via protein denaturation induced by heat or chaotropic salts, typically urea or guanidine hydrochloride, followed by refolding. Yet, TM proteins typically aggregate at increasing temperatures, preventing refolding, and as for in vitro studies, isolated TM proteins are analyzed when incorporated into a lipid bilayer or a detergent micelle, where individual TM helices are shielded from the aqueous environment. Consequently, whereas urea or GdnHCl can unfold extramembranous domains to disrupt TM contacts, the properties of the intimate solvent (here, the membrane or detergent micelles) have to be modified. Cys-labeling kinetics of membrane protein GlpG: a role for specific SDS binding and micelle changes?Otzen et al.Biophysical JournalAugust 6, 2021In BriefEmpirically, α-helical membrane protein folding stability in surfactant micelles can be tuned by varying the mole fraction MFSDS of anionic (sodium dodecyl sulfate (SDS)) relative to nonionic (e.g., dodecyl maltoside (DDM)) surfactant, but we lack a satisfying physical explanation of this phenomenon. Cysteine labeling (CL) has thus far only been used to study the topology of membrane proteins, not their stability or folding behavior. Here, we use CL to investigate membrane protein folding in mixed DDM-SDS micelles. Full-Text PDF

Odorant binding proteins (OBPs) are small proteins, some of which bind odorants with high specificity. OBPs are relatively easy to produce and show a pronounced stability toward thermal and chemical denaturation. This high stability renders OBPs attractive candidates for the development of odorant detections systems. Unfortunately, binding of odorants is not easy to quantify due to lack of spectroscopic signals upon binding. Therefore, a possible approach to detect binding is to employ the shift in thermal or chemical stability upon ligand-protein interaction. Being a rather indirect approach, the experimental setup should be done with care. Here, the experimental results on stability of OBPs are summarized and issues which should be considered when performing stability experiments are discussed.

ABSTRACTThe 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.