Structural basis for Vipp1 membrane binding: from loose coats and carpets to ring and rod assemblies.

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.

Dual Centrifugation-based Screening for pH-responsive Liposomes.

In liposomal drug delivery development, the delicate balance of membrane stability is a major challenge to prevent leakage (during shelf-life and blood circulation), and to ensure efficient payload release at the therapeutic destination. Our composite screening approach uses the processing by dual centrifugation technique to speed up the identification of de novo formulations of intermediate membrane stability. By screening binary lipid combinations at systemically varied ratios we highlight liposomal formulations of intermediate stability, what we termed "the edge of stability", requiring moderate stimuli for destabilization. Supplementation with a pH-sensitive cholesterol derivative (to obtain acid labile liposomes) and renewed assessment with cargo load led to the discovery of three formulations with sufficient shelf-life stability, acceptable cargo retention and efficient pH-responsive cargo release in vitro. The "lead candidates" exhibited promising in cellulo uptake with increased intracellular cargo release and revealed in vivo performance advantages compared to a control liposome. Our approach filters lipid compositions on "the edge of stability" that were introduced with a pH-sensitive cholesterol derivate leading pH-responsive liposomes, out of a multidimensional parameter space. Their discovery by rational approaches would have been highly unlikely, thus highlighting the potential of our screening approach.

Structural basis for GTPase activity and conformational changes of the bacterial dynamin-like protein SynDLP.

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.

Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies.

Eukaryotic members of the endosome sorting complex required for transport-III (ESCRT-III) family have been shown to form diverse higher-order assemblies. The bacterial phage shock protein A (PspA) has been identified as a member of the ESCRT-III superfamily, and PspA homo-oligomerizes to form rod-shaped assemblies. As observed for eukaryotic ESCRT-III, PspA forms tubular assemblies of varying diameters. Using electron cryo-electron microscopy, we determined 61 Synechocystis PspA structures and observed in molecular detail how the structural plasticity of PspA rods is mediated by conformational changes at three hinge regions in the monomer and by the fixed and changing molecular contacts between protomers. Moreover, we reduced and increased the structural plasticity of PspA rods by removing the loop connecting helices α3/α4 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 members.

Mg2+ limitation leads to a decrease in chlorophyll, resulting in an unbalanced photosynthetic apparatus in the cyanobacterium Synechocytis sp. PCC6803.

Mg2+, the most abundant divalent cation in living cells, plays a pivotal role in numerous enzymatic reactions and is of particular importance for organisms performing oxygenic photosynthesis. Its significance extends beyond serving as the central ion of the chlorophyll molecule, as it also acts as a counterion during the light reaction to balance the proton gradient across the thylakoid membranes. In this study, we investigated the effects of Mg2+ limitation on the physiology of the well-known model microorganism Synechocystis sp. PCC6803. Our findings reveal that Mg2+ deficiency triggers both morphological and functional changes. As seen in other oxygenic photosynthetic organisms, Mg2+ deficiency led to a decrease in cellular chlorophyll concentration. Moreover, the PSI-to-PSII ratio decreased, impacting the photosynthetic efficiency of the cell. In line with this, Mg2+ deficiency led to a change in the proton gradient built up across the thylakoid membrane upon illumination.

Ultrasound-guided determination demonstrates influence of age, sex and type of sport on medial femoral condyle cartilage thickness in children and adolescents.

PURPOSE: To analyse the reliability of ultrasound-guided measurement of the cartilage thickness at the medial femoral condyle in athletically active children and adolescents before and after mechanical load in relation to age, sex and type of sport. METHODS: Three successive measurements were performed in 157 participants (median/min-max age: 13.1/6.0-18.0 years, 106 males) before and after mechanical load by squats at the same site of the medial femoral condyle by defined transducer positioning. Test-retest reliability was examined using Cronbach's α $\alpha $ calculation. Differences in cartilage thickness were analysed with respect to age, sex and type of practiced sports, respectively. RESULTS: Excellent reliability was achieved both before and after mechanical load by 30 squats with a median cartilage thickness of 1.9 mm (range: 0.5-4.8 mm) before and 1.9 mm (0.4-4.6 mm) after mechanical load. Male cartilages were thicker (p < 0.01) before (median: 2.0 mm) and after (2.0 mm) load when compared to female cartilage (before: 1.6 mm; after: 1.7 mm). Median cartilage thickness was about three times higher in karate athletes (before: 2.3 mm; after: 2.4 mm) than in sports shooters (0.7; 0.7 mm). Cartilage thickness in track and field athletes, handball players and soccer players were found to lay in-between. Sport type related thickness changes after mechanical load were not significant. CONCLUSION: Medial femoral condyle cartilage thickness in childhood correlates with age, sex and practiced type of sports. Ultrasound is a reliable and simple, pain-free approach to evaluate the cartilage thickness in children and adolescents. LEVEL OF EVIDENCE: Level III.

Conserved structures of ESCRT-III superfamily members across domains of life.

Structural and evolutionary studies of cyanobacterial phage shock protein A (PspA) and inner membrane-associated protein of 30 kDa (IM30) have revealed that these proteins belong to the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, which is conserved across all three domains of life. PspA and IM30 share secondary and tertiary structures with eukaryotic ESCRT-III proteins, whilst also oligomerizing via conserved interactions. Here, we examine the structures of bacterial ESCRT-III-like proteins and compare the monomeric and oligomerized forms with their eukaryotic counterparts. We discuss conserved interactions used for self-assembly and highlight key hinge regions that mediate oligomer ultrastructure versatility. Finally, we address the differences in nomenclature assigned to equivalent structural motifs in both the bacterial and eukaryotic fields and suggest a common nomenclature applicable across the ESCRT-III superfamily.

SynDLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features.

Dynamin-like proteins are membrane remodeling GTPases with well-understood functions in eukaryotic cells. However, bacterial dynamin-like proteins are still poorly investigated. SynDLP, the dynamin-like protein of the cyanobacterium Synechocystis sp. PCC 6803, forms ordered oligomers in solution. The 3.7 Å resolution cryo-EM structure of SynDLP oligomers reveals the presence of oligomeric stalk interfaces typical for eukaryotic dynamin-like proteins. The bundle signaling element domain shows distinct features, such as an intramolecular disulfide bridge that affects the GTPase activity, or an expanded intermolecular interface with the GTPase domain. In addition to typical GD-GD contacts, such atypical GTPase domain interfaces might be a GTPase activity regulating tool in oligomerized SynDLP. Furthermore, we show that SynDLP interacts with and intercalates into membranes containing negatively charged thylakoid membrane lipids independent of nucleotides. The structural characteristics of SynDLP oligomers suggest it to be the closest known bacterial ancestor of eukaryotic dynamin.

Unfolding Individual Domains of BmrA, a Bacterial ABC Transporter Involved in Multidrug Resistance.

The folding and stability of proteins are often studied via unfolding (and refolding) a protein with urea. Yet, in the case of membrane integral protein domains, which are shielded by a membrane or a membrane mimetic, urea generally does not induce unfolding. However, the unfolding of α-helical membrane proteins may be induced by the addition of sodium dodecyl sulfate (SDS). When protein unfolding is followed via monitoring changes in Trp fluorescence characteristics, the contributions of individual Trp residues often cannot be disentangled, and, consequently, the folding and stability of the individual domains of a multi-domain membrane protein cannot be studied. In this study, the unfolding of the homodimeric bacterial ATP-binding cassette (ABC) transporter Bacillus multidrug resistance ATP (BmrA), which comprises a transmembrane domain and a cytosolic nucleotide-binding domain, was investigated. To study the stability of individual BmrA domains in the context of the full-length protein, the individual domains were silenced by mutating the existent Trps. The SDS-induced unfolding of the corresponding constructs was compared to the (un)folding characteristics of the wild-type (wt) protein and isolated domains. The full-length variants BmrAW413Y and BmrAW104YW164A were able to mirror the changes observed with the isolated domains; thus, these variants allowed for the study of the unfolding and thermodynamic stability of mutated domains in the context of full-length BmrA.

Cyanobacterial membrane dynamics in the light of eukaryotic principles.

Intracellular compartmentalization is a hallmark of eukaryotic cells. Dynamic membrane remodeling, involving membrane fission/fusion events, clearly is crucial for cell viability and function, as well as membrane stabilization and/or repair, e.g., during or after injury. In recent decades, several proteins involved in membrane stabilization and/or dynamic membrane remodeling have been identified and described in eukaryotes. Yet, while typically not having a cellular organization as complex as eukaryotes, also bacteria can contain extra internal membrane systems besides the cytoplasmic membranes (CMs). Thus, also in bacteria mechanisms must have evolved to stabilize membranes and/or trigger dynamic membrane remodeling processes. In fact, in recent years proteins, which were initially defined being eukaryotic inventions, have been recognized also in bacteria, and likely these proteins shape membranes also in these organisms. One example of a complex prokaryotic inner membrane system is the thylakoid membrane (TM) of cyanobacteria, which contains the complexes of the photosynthesis light reaction. Cyanobacteria are evolutionary closely related to chloroplasts, and extensive remodeling of the internal membrane systems has been observed in chloroplasts and cyanobacteria during membrane biogenesis and/or at changing light conditions. We here discuss common principles guiding eukaryotic and prokaryotic membrane dynamics and the proteins involved, with a special focus on the dynamics of the cyanobacterial TMs and CMs.

Hydrophobic mismatch and sequence specificity compete when transmembrane helix-helix interactions are measured with the TOXCAT assay.

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.

The Peripherally Membrane-attached Protein MbFACL6 of Mycobacterium tuberculosis Activates a Broad Spectrum of Substrates.

The infectious disease tuberculosis is one of the fifteen most common causes of death worldwide (according to the WHO). About every fourth person is infected with the main causative agent Mycobacterium tuberculosis (Mb). A characteristic of the pathogen is its entrance into a dormant state in which a phenotypic antibiotic resistance is achieved. To target resistant strains, novel dormancy-specific targets are very promising. Such a possible target is the Mb "fatty acid-CoA ligase 6" (MbFACL6), which activates fatty acids and thereby modulates the accumulation of triacylglycerol-containing lipid droplets that are used by Mb as an energy source during dormancy. We investigated the membrane association of MbFACL6 in E. coli and its specific activity towards different substrates after establishing a novel MbFACL6 activity assay. Despite a high homology to the mammalian family of fatty acid transport proteins, which are typically transmembrane proteins, our results indicate that MbFACL6 is a peripheral membrane-attached protein. Furthermore, MbFACL6 tolerates a broad spectrum of substrates including saturated and unsaturated fatty acids (C12-C20), some cholic acid derivatives, and even synthetic fatty acids, such as 9(E)-nitrooleicacid. Therefore, the substrate selectivity of MbFACL6 appears to be much broader than previously assumed.

Membrane destabilization and pore formation induced by the Synechocystis IM30 protein.

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.

CLiB - a novel cardiolipin-binder isolated via data-driven and in vitro screening.

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.

Human Claudin-7 cis-Interactions Are Not Crucial for Membrane-Membrane (Trans-) Interactions.

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.

Data-driven discovery of cardiolipin-selective small molecules by computational active learning.

Subtle variations in the lipid composition of mitochondrial membranes can have a profound impact on mitochondrial function. The inner mitochondrial membrane contains the phospholipid cardiolipin, which has been demonstrated to act as a biomarker for a number of diverse pathologies. Small molecule dyes capable of selectively partitioning into cardiolipin membranes enable visualization and quantification of the cardiolipin content. Here we present a data-driven approach that combines a deep learning-enabled active learning workflow with coarse-grained molecular dynamics simulations and alchemical free energy calculations to discover small organic compounds able to selectively permeate cardiolipin-containing membranes. By employing transferable coarse-grained models we efficiently navigate the all-atom design space corresponding to small organic molecules with molecular weight less than ≈500 Da. After direct simulation of only 0.42% of our coarse-grained search space we identify molecules with considerably increased levels of cardiolipin selectivity compared to a widely used cardiolipin probe 10-N-nonyl acridine orange. Our accumulated simulation data enables us to derive interpretable design rules linking coarse-grained structure to cardiolipin selectivity. The findings are corroborated by fluorescence anisotropy measurements of two compounds conforming to our defined design rules. Our findings highlight the potential of coarse-grained representations and multiscale modelling for materials discovery and design.

Increased stability of the TM helix oligomer abrogates the apoptotic activity of the human Fas receptor.

Human death receptors control apoptotic events during cell differentiation, cell homeostasis and the elimination of damaged or infected cells. Receptor activation involves ligand-induced structural reorganizations of preformed receptor trimers. Here we show that the death receptor transmembrane domains only have a weak intrinsic tendency to homo-oligomerize within a membrane, and thus these domains potentially do not significantly contribute to receptor trimerization. However, mutation of Pro183 in the human CD95/Fas receptor transmembrane helix results in a dramatically increased interaction propensity, as shown by genetic assays. The increased interaction of the transmembrane domain is coupled with a decreased ligand-sensitivity of cells expressing the Fas receptor, and thus in a decreased number of apoptotic events. Mutation of Pro183 likely results in a substantial rearrangement of the self-associated Fas receptor transmembrane trimer, which likely abolishes further signaling of the apoptotic signal but may activate other signaling pathways. Our study shows that formation of a stable Fas receptor transmembrane helix oligomer does not per se result in receptor activation.

Myristic Acid Inhibits the Activity of the Bacterial ABC Transporter BmrA.

ATP-binding cassette (ABC) transporters are conserved in all kingdoms of life, where they transport substrates against a concentration gradient across membranes. Some ABC transporters are known to cause multidrug resistances in humans and are able to transport chemotherapeutics across cellular membranes. Similarly, BmrA, the ABC transporter of Bacillus subtilis, is involved in excretion of certain antibiotics out of bacterial cells. Screening of extract libraries isolated from fungi revealed that the C14 fatty acid myristic acid has an inhibitory effect on the BmrA ATPase as well as the transport activity. Thus, a natural membrane constituent inhibits the BmrA activity, a finding with physiological consequences as to the activity and regulation of ABC transporter activities in biological membranes.

Quantitative characterization of tetraspanin 8 homointeractions in the plasma membrane.

The spatial distribution of proteins in cell membranes is crucial for signal transduction, cell communication and membrane trafficking. Members of the Tetraspanin family organize functional protein clusters within the plasma membrane into so-called Tetraspanin-enriched microdomains (TEMs). Direct interactions between Tetraspanins are believed to be important for this organization. However, studies thus far have utilized mainly co-immunoprecipitation methods that cannot distinguish between direct and indirect, through common partners, interactions. Here we study Tetraspanin 8 homointeractions in living cells via quantitative fluorescence microscopy. We demonstrate that Tetraspanin 8 exists in a monomer-dimer equilibrium in the plasma membrane. Tetraspanin 8 dimerization is described by a high dissociation constant (Kd = 14 700 ± 1100 Tspan8/µm2), one of the highest dissociation constants measured for membrane proteins in live cells. We propose that this high dissociation constant, and thus the short lifetime of the Tetraspanin 8 dimer, is critical for Tetraspanin 8 functioning as a master regulator of cell signaling.