A metastasis-associated Pannexin1 mutant (Panx1 1-89 ) forms a minimalist ATP release channel.

A truncated form of the ATP release channel pannexin 1 (Panx1), Panx1 1-89 , is enriched in metastatic breast cancer cells and has been proposed to mediate metastatic cell survival by increasing ATP release through mechanosensitive Panx1 channels. However, whether Panx1 1-89 on its own (without the presence of wtPanx1) mediates ATP release has not been tested. Here, we show that Panx1 1-89 by itself can form a constitutively active membrane channel, capable of releasing ATP even in the absence of wild type Panx1. Our biophysical characterization reveals that most basic structure-function features of the channel pore are conserved in the truncated Panx1 1-89 peptide. Thus, augmenting extracellular potassium ion concentrations enhances Panx1 1-89 -mediated conductance. Moreover, despite the severe truncation, Panx1 1-89 retains the sensitivity to most of wtPanx1 channel inhibitors and can thus be targeted. Therefore, Panx1 blockers have the potential to be of therapeutic value to combat metastatic cell survival. Our study not only elucidates a mechanism for ATP release from cancer cells, but it also supports that the Panx1 1-89 mutant should facilitate structure-function analysis of Panx1 channels.

Silk Assembly against Hydrophobic Surfaces─Modeling and Imaging of Formation of Nanofibrils.

A detailed insight about the molecular organization behind spider silk assembly is valuable for the decoding of the unique properties of silk. The recombinant partial spider silk protein 4RepCT contains four poly-alanine/glycine-rich repeats followed by an amphiphilic C-terminal domain and has shown the capacity to self-assemble into fibrils on hydrophobic surfaces. We herein use molecular dynamic simulations to address the structure of 4RepCT and its different parts on hydrophobic versus hydrophilic surfaces. When 4RepCT is placed in a wing arrangement model and periodically repeated on a hydrophobic surface, ß-sheet structures of the poly-alanine repeats are preserved, while the CT part is settled on top, presenting a fibril with a height of ∼7 nm and a width of ∼11 nm. Both atomic force microscopy and cryo-electron microscopy imaging support this model as a possible fibril formation on hydrophobic surfaces. These results contribute to the understanding of silk assembly and alignment mechanism onto hydrophobic surfaces.

Mechanism of action of IC 100, a humanized IgG4 monoclonal antibody targeting apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC).

Inflammasomes are multiprotein complexes of the innate immune response that recognize a diverse range of intracellular sensors of infection or cell damage and recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into an inflammasome signaling complex. The recruitment, polymerization and cross-linking of ASC is upstream of caspase-1 activation and interleukin-1ß release. Here we provide evidence that IC 100, a humanized IgG4κ monoclonal antibody against ASC, is internalized into the cell and localizes with endosomes, while another part is recycled and redistributed out of the cell. IC 100 binds intracellular ASC and blocks interleukin-1ß release in a human whole blood cell inflammasome assay. In vitro studies demonstrate that IC 100 interferes with ASC polymerization and assembly of ASC specks. In vivo bioluminescence imaging showed that IC 100 has broad tissue distribution, crosses the blood brain barrier, and readily penetrates the brain and spinal cord parenchyma. Confocal microscopy of fluorescent-labeled IC 100 revealed that IC 100 is rapidly taken up by macrophages via a mechanism utilizing the Fc region of IC 100. Coimmunoprecipitation experiments and confocal immunohistochemistry showed that IC 100 binds to ASC and to the atypical antibody receptor Tripartite motif-containing protein-21 (TRIM21). In A549 WT and TRIM21 KO cells treated with either IC 100 or IgG4κ isotype control, the levels of intracellular IC 100 were higher than in the IgG4κ-treated controls at 2 hours, 1 day and 3 days after administration, indicating that IC 100 escapes degradation by the proteasome. Lastly, electron microscopy studies demonstrate that IC 100 binds to ASC filaments and alters the architecture of ASC filaments. Thus, IC 100 readily penetrates a variety of cell types, and it binds to intracellular ASC, but it is not degraded by the TRIM21 antibody-dependent intracellular neutralization pathway.

The long noncoding RNA ADIPINT regulates human adipocyte metabolism via pyruvate carboxylase.

The pleiotropic function of long noncoding RNAs is well recognized, but their direct role in governing metabolic homeostasis is less understood. Here, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase, a pivotal enzyme in energy metabolism. We developed an approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation, which identifies that ADIPINT binds to pyruvate carboxylase and validated the interaction with electron microscopy. ADIPINT knockdown alters the interactome and decreases the abundance and enzymatic activity of pyruvate carboxylase in the mitochondria. Reduced ADIPINT or pyruvate carboxylase expression lowers adipocyte lipid synthesis, breakdown, and lipid content. In human white adipose tissue, ADIPINT expression is increased in obesity and linked to fat cell size, adipose insulin resistance, and pyruvate carboxylase activity. Thus, we identify ADIPINT as a regulator of lipid metabolism in human white adipocytes, which at least in part is mediated through its interaction with pyruvate carboxylase.

A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo.

Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34+ hematopoietic stem cells from peripheral blood stem cell grafts and CD34+ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34+ blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.

Optimizing purification of the peripheral membrane protein FAM92A1 fused to a modified spidroin tag.

Cryo-electron microscopy has revolutionized structural biology. In particular structures of proteins at the membrane interface have been a major contribution of cryoEM. Yet, visualization and characterization of peripheral membrane proteins remains challenging; mostly because there is no unified purification strategy for these proteins. FAM92A1 is a novel peripheral membrane protein that binds to the mitochondrial inner membrane. There, FAM92A1 dimers bind to the membrane and play an essential role in regulating the mitochondrial ultrastructure. Curiously, FAM92A1 has also an important function in ciliogenesis. FAM92A1 is part of the membrane bending Bin1/Amphiphsyin/RVS (BAR) domain protein family. Currently, there is no structure of FAM92A1, mostly because FAM92A1 is unstable and insoluble at high concentrations, like many BAR domain proteins. Yet, pure and concentrated protein is a necessity for screening to generate samples suitable for structure determination. Here, we present an optimized purification and expression strategy for dimeric FAM92A1. To our knowledge, we are the first to use the spidroin tag NT* to successfully purify a peripheral membrane protein. Our results show that NT* not only increases solubility but stabilizes FAM92A1 as a dimer. FAM92A1 fused to NT* is active because it is able to efficiently bend membranes. Taken together, our strategy indicates that this is a possible avenue to express and purify other challenging BAR domain proteins.

Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces.

Over the last few years, cryo electron microscopy has become the most important method in structural biology. While 80% of deposited maps are from single particle analysis, electron tomography has grown to become the second most important method. In particular sub-tomogram averaging has matured as a method, delivering structures between 2 and 5 Å from complexes in cells as well as in vitro complexes. While this resolution range is not standard, novel developments point toward a promising future. Here, we provide a guide for the workflow from sample to structure to gain insight into this emerging field.

CryoEM reveals BIN1 (isoform 8) does not bind to single actin filaments in vitro.

Cells change their appearance by a concerted action of the cytoskeleton and the plasma membrane. The machinery that bends the membrane includes Bin/Amphiphysin/Rvs (BAR) domain proteins. Recently BAR domain proteins garnered attention as actin regulators, either by recruiting actin regulating proteins or through binding to actin directly. BIN1 (an important protein in Alzheimer's Disease, heart disease and cancer) is one of the few BAR proteins that bind to actin directly. Here, we imaged a complex of BIN1 and actin with cryoEM. Our results reveal that BIN1 cannot be found on single actin filaments.

Structure versus function: Are new conformations of pannexin 1 yet to be resolved?

Pannexin 1 (Panx1) plays a decisive role in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It is widely accepted that Panx1 exerts its role in the context of purinergic signaling by providing a transmembrane pathway for ATP. However, under certain conditions, Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability. A recent flurry of publications has provided structural information about the Panx1 channel. However, while these structures are consistent with a chloride selective channel, none show a conformation with strong support for the ATP release function of Panx1. In this Viewpoint, we critically assess the existing evidence for the function and structure of the Panx1 channel and conclude that the structure corresponding to the ATP permeation pathway is yet to be determined. We also list a set of additional topics needing attention and propose ways to attain the large-pore, ATP-permeable conformation of the Panx1 channel.

Structural basis for SARM1 inhibition and activation under energetic stress.

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1's own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.

Cells Control BIN1-Mediated Membrane Tubulation by Altering the Membrane Charge.

The Bridging integrator 1 (BIN1)/Amphiphysin/Rvs (BAR) protein family is an essential part of the cell's machinery to bend membranes. BIN1 is a muscle-enriched BAR protein with an established role in muscle development and skeletal myopathies. Here, we demonstrate that BIN1, on its own, is able to form complex interconnected tubular systems in vitro, reminiscent of t-tubule system in muscle cells. We further describe how BIN1's electrostatic interactions regulate membrane bending: the ratio of negatively charged lipids in the bilayer altered membrane bending and binding properties of BIN1 and so did the manipulation of BIN1's surface charge. We show that the electrostatically mediated BIN1 membrane binding depended on the membrane curvature-it was less affected in liposomes with high curvature. Curiously, BIN1 membrane binding and bending was diminished in cells where the membrane's charge was experimentally reduced. Membrane bending was also reduced in BIN1 mutants where negative or positive charges in the BAR domain have been eliminated. This phenotype, characteristic of BIN1 mutants linked to myopathies, was rescued when the membrane charge was made more negative. The latter findings also show that cells can control tubulation at their membranes by simply altering the membrane charge and through it, the recruitment of BAR proteins and their interaction partners (e.g. dynamin).

Structural Evidence for an Octameric Ring Arrangement of SARM1.

SARM1 induces axonal degeneration in response to various insults and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD+ to ADPR (ADP Ribose) products by the SARM1's TIR domain. Therefore, inhibition of either SAM or TIR functions may constitute an effective therapeutic strategy. However, there is currently no SARM1-directed therapeutic approach available because of an insufficient structural and mechanistic understanding of this protein. In this study, we found that SARM1 assembles into an octameric ring. This arrangement was not described before in other SAM proteins, but is reminiscent of the apoptosome and inflammasome-well-known apoptotic ring-like oligomers. We show that both SARM1 and the isolated tandem SAM1-2 domains form octamers in solution, and electron microscopy analysis reveals an octameric ring of SARM1. We determined the crystal structure of SAM1-2 and found that it also forms a closed octameric ring in the crystal lattice. The SAM1-2 ring interactions are mediated by complementing "lock and key" hydrophobic grooves and inserts and electrostatic charges between the neighboring protomers. We have mutated several interacting SAM1-2 interfaces and measured how these mutations affect SARM1 apoptotic activity in cultured cells, and in this way identified critical oligomerization sites that facilitate cell death. These results highlight the importance of oligomerization for SARM1 function and reveal critical epitopes for future targeted drug development.

Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity.

Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.

Regulated protein turnover: snapshots of the proteasome in action.

The ubiquitin proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and misfolded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures provide some surprising insights into the functional mechanism of the proteasome and will give invaluable guidance for genetic and biochemical studies of this key regulatory system.

Protein-mediated transformation of lipid vesicles into tubular networks.

Key cellular processes are frequently accompanied by protein-facilitated shape changes in the plasma membrane. N-BAR-domain protein modules generate curvature by means of complex interactions with the membrane surface. The way they assemble and the mechanism by which they operate are largely dependent on their binding density. Although the mechanism at lower densities has recently begun to emerge, how membrane scaffolds form at high densities remains unclear. By combining electron microscopy and multiscale simulations, we show that N-BAR proteins at high densities can transform a lipid vesicle into a 3D tubular network. We show that this process is a consequence of excess adhesive energy combined with the local stiffening of the membrane, which occurs in a narrow range of mechanical properties of both the membrane and the protein. We show that lipid diffusion is significantly reduced by protein binding at this density regime and even more in areas of high Gaussian curvature, indicating a potential effect on molecular transport in cells. Finally, we reveal that the breaking of the bilayer topology is accompanied by the nematic arrangement of the protein on the surface, a structural motif that likely drives the formation of reticular structures in living cells.

Understanding the role of amphipathic helices in N-BAR domain driven membrane remodeling.

Endophilin N-BAR (N-terminal helix and Bin/amphiphysin/Rvs) domain tubulates and vesiculates lipid membranes in vitro via its crescent-shaped dimer and four amphipathic helices that penetrate into membranes as wedges. Like F-BAR domains, endophilin N-BAR also forms a scaffold on membrane tubes. Unlike F-BARs, endophilin N-BARs have N-terminal H0 amphipathic helices that are proposed to interact with other N-BARs in oligomer lattices. Recent cryo-electron microscopy reconstructions shed light on the organization of the N-BAR lattice coats on a nanometer scale. However, because of the resolution of the reconstructions, the precise positioning of the amphipathic helices is still ambiguous. In this work, we applied a coarse-grained model to study various membrane remodeling scenarios induced by endophilin N-BARs. We found that H0 helices of N-BARs prefer to align in an antiparallel manner at two ends of the protein to form a stable lattice. The deletion of H0 helices causes disruption of the lattice. In addition, we analyzed the persistence lengths of the protein-coated tubes and found that the stiffness of endophilin N-BAR-coated tubules qualitatively agrees with previous experimental work studying N-BAR-coated tubules. Large-scale simulations on membrane liposomes revealed a systematic relation between H0 helix density and local membrane curvature fluctuations. The data also suggest that the H0 helix is required for BARs to form organized structures on the liposome, further illustrating its important function.

Membrane curvature and its generation by BAR proteins.

Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the Bin/amphiphysin/Rvs (BAR) domain proteins. Many open questions remain about the mechanisms by which membrane-bending proteins function. Addressing this shortfall, recent structures of BAR protein:membrane complexes support existing mechanistic models, but also produced novel insights into how BAR domain proteins sense, stabilize, and generate curvature. Here we review these recent findings, focusing on how BAR proteins interact with the membrane, and how the resulting scaffold structures might aid the recruitment of other proteins to the sites where membranes are bent.

Structural basis of membrane bending by the N-BAR protein endophilin.

Functioning as key players in cellular regulation of membrane curvature, BAR domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic and that the N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin's SH3 domains, suggesting that the spatial presentation of SH3 domains, rather than affinity, governs the recruitment of downstream interaction partners.

New insights into BAR domain-induced membrane remodeling.

Mesoscopic simulations and electron microscopy of N-BAR domain-induced liposome remodeling are used to characterize the process of liposome tubulation and vesiculation. The overall process of membrane remodeling is found to involve complex couplings among the N-BAR protein density, the degree of N-BAR oligomerization, and the membrane density. A comparison of complex remodeled liposome structures from mesoscopic simulations with those measured by electron microscopy experiments suggests that the process of membrane remodeling can be described via an appropriate mesoscopic free energy framework. Liposome remodeling more representative of F-BAR domains is also presented within the mesoscopic simulation framework.

Two conformational changes are associated with glutamate translocation by the glutamate transporter EAAC1.

Glutamate is transported across membranes by means of a carrier mechanism that is thought to require conformational changes of the transport protein. In this work, we have determined the thermodynamic parameters of glutamate and the Na+ binding steps to their extracellular binding sites along with the activation parameters of rapid, glutamate-induced processes in the transport cycle by analyzing the temperature dependence of glutamate transport at steady state and pre-steady state. Our results suggest that glutamate binding to the transporter is driven by a negative reaction enthalpy (DeltaH0 = -33 kJ/mol), whereas the tighter binding of the non-transportable inhibitor TBOA is caused by an additional increase in entropy. Processes linked to the binding of glutamate and Na+ to the transporter are associated with low activation barriers, indicative of diffusion-controlled reactions. The activation enthalpies of two processes in the glutamate translocation branch of the transport cycle were DeltaH++ = 95 kJ/mol and DeltaH++ = 120 kJ/mol, respectively. Such large values of DeltaH++ suggest that these processes are rate-limited by conformational changes of the transporter. We also found a large activation barrier for steady-state glutamate transport, which is rate-limited by the K+-dependent relocation of the empty transporter. Together, these results suggest that two conformational changes accompany glutamate translocation and at least one conformational change accompanies the relocation of the empty transporter. We interpret the data with an alternating access model that includes the closing and opening of an extracellular and an intracellular gate, respectively, in analogy to a hypothetical model proposed previously on the basis of the crystal structure of the bacterial glutamate transporter GltPh.