Structural titration reveals Ca2+-dependent conformational landscape of the IP3 receptor.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are endoplasmic reticulum Ca2+ channels whose biphasic dependence on cytosolic Ca2+ gives rise to Ca2+ oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IP3R-mediated Ca2+ responses, the structural underpinnings of the biphasic Ca2+ dependence that underlies Ca2+ oscillations are incompletely understood. Here, we collect cryo-EM images of an IP3R with Ca2+ concentrations spanning five orders of magnitude. Unbiased image analysis reveals that Ca2+ binding does not explicitly induce conformational changes but rather biases a complex conformational landscape consisting of resting, preactivated, activated, and inhibited states. Using particle counts as a proxy for relative conformational free energy, we demonstrate that Ca2+ binding at a high-affinity site allows IP3Rs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca2+ concentrations, IP3Rs preferentially enter an inhibited state stabilized by a second, low-affinity Ca2+ binding site. Together, these studies provide a mechanistic basis for the biphasic Ca2+-dependence of IP3R channel activity.

A pentameric TRPV3 channel with a dilated pore.

Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets1-5. More than 210 structures from more than 20 different TRP channels have been determined, and all are tetramers4. Despite this wealth of structures, many aspects concerning TRPV channels remain poorly understood, including the pore-dilation phenomenon, whereby prolonged activation leads to increased conductance, permeability to large ions and loss of rectification6,7. Here, we used high-speed atomic force microscopy (HS-AFM) to analyse membrane-embedded TRPV3 at the single-molecule level and discovered a pentameric state. HS-AFM dynamic imaging revealed transience and reversibility of the pentamer in dynamic equilibrium with the canonical tetramer through membrane diffusive protomer exchange. The pentamer population increased upon diphenylboronic anhydride (DPBA) addition, an agonist that has been shown to induce TRPV3 pore dilation. On the basis of these findings, we designed a protein production and data analysis pipeline that resulted in a cryogenic-electron microscopy structure of the TRPV3 pentamer, showing an enlarged pore compared to the tetramer. The slow kinetics to enter and exit the pentameric state, the increased pentamer formation upon DPBA addition and the enlarged pore indicate that the pentamer represents the structural correlate of pore dilation. We thus show membrane diffusive protomer exchange as an additional mechanism for structural changes and conformational variability. Overall, we provide structural evidence for a non-canonical pentameric TRP-channel assembly, laying the foundation for new directions in TRP channel research.

Structures of ß1-adrenergic receptor in complex with Gs and ligands of different efficacies.

G-protein-coupled receptors (GPCRs) receive signals from ligands with different efficacies, and transduce to heterotrimeric G-proteins to generate different degrees of physiological responses. Previous studies revealed how ligands with different efficacies activate GPCRs. Here, we investigate how a GPCR activates G-proteins upon binding ligands with different efficacies. We report the cryo-EM structures of ß1-adrenergic receptor (ß1-AR) in complex with Gs (GαsGß1Gγ2) and a partial agonist or a very weak partial agonist, and compare them to the ß1-AR-Gs structure in complex with a full agonist. Analyses reveal similar overall complex architecture, with local conformational differences. Cellular functional studies with mutations of ß1-AR residues show effects on the cellular signaling from ß1-AR to the cAMP response initiated by the three different ligands, with residue-specific functional differences. Biochemical investigations uncover that the intermediate state complex comprising ß1-AR and nucleotide-free Gs is more stable when binding a full agonist than a partial agonist. Molecular dynamics simulations support the local conformational flexibilities and different stabilities among the three complexes. These data provide insights into the ligand efficacy in the activation of GPCRs and G-proteins.

Allosteric interactions prime androgen receptor dimerization and activation.

The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.

Differential activation mechanisms of lipid GPCRs by lysophosphatidic acid and sphingosine 1-phosphate.

Lysophospholipids are bioactive lipids and can signal through G-protein-coupled receptors (GPCRs). The best studied lysophospholipids are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). The mechanisms of lysophospholipid recognition by an active GPCR, and the activations of lysophospholipid GPCR-G-protein complexes remain unclear. Here we report single-particle cryo-EM structures of human S1P receptor 1 (S1P1) and heterotrimeric Gi complexes formed with bound S1P or the multiple sclerosis (MS) treatment drug Siponimod, as well as human LPA receptor 1 (LPA1) and Gi complexes in the presence of LPA. Our structural and functional data provide insights into how LPA and S1P adopt different conformations to interact with their cognate GPCRs, the selectivity of the homologous lipid GPCRs for S1P versus LPA, and the different activation mechanisms of these GPCRs by LPA and S1P. Our studies also reveal specific optimization strategies to improve the MS-treating S1P1-targeting drugs.

Structural basis and mechanism of activation of two different families of G proteins by the same GPCR.

The ß1-adrenergic receptor (ß1-AR) can activate two families of G proteins. When coupled to Gs, ß1-AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey ß1-AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gαi and Gαs interact similarly with ß1-AR, the overall interacting modes between ß1-AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by ß1-AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4).

Structural Basis of the Activation of Heterotrimeric Gs-Protein by Isoproterenol-Bound ß1-Adrenergic Receptor.

Cardiac disease remains the leading cause of morbidity and mortality worldwide. The ß1-adrenergic receptor (ß1-AR) is a major regulator of cardiac functions and is downregulated in the majority of heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by ß1-ARs, leading to increased heart rate and contractility. Here, we use cryo-electron microscopy and functional studies to investigate the molecular mechanism by which ß1-AR activates Gs. We find that the tilting of α5-helix breaks a hydrogen bond between the sidechain of His373 in the C-terminal α5-helix and the backbone carbonyl of Arg38 in the N-terminal αN-helix of Gαs. Together with the disruption of another interacting network involving Gln59 in the α1-helix, Ala352 in the ß6-α5 loop, and Thr355 in the α5-helix, these conformational changes might lead to the deformation of the GDP-binding pocket. Our data provide molecular insights into the activation of G-proteins by G-protein-coupled receptors.

Gating and selectivity mechanisms for the lysosomal K+ channel TMEM175.

Transmembrane protein 175 (TMEM175) is a K+-selective ion channel expressed in lysosomal membranes, where it establishes a membrane potential essential for lysosomal function and its dysregulation is associated with the development of Parkinson's Disease. TMEM175 is evolutionarily distinct from all known channels, predicting novel ion-selectivity and gating mechanisms. Here we present cryo-EM structures of human TMEM175 in open and closed conformations, enabled by resolutions up to 2.6 Å. Human TMEM175 adopts a homodimeric architecture with a central ion-conduction pore lined by the side chains of the pore-lining helices. Conserved isoleucine residues in the center of the pore serve as the gate in the closed conformation. In the widened channel in the open conformation, these same residues establish a constriction essential for K+ selectivity. These studies reveal the mechanisms of permeation, selectivity and gating and lay the groundwork for understanding the role of TMEM175 in lysosomal function.

Potential Hydrodynamic Cytoplasmic Transfer between Mammalian Cells: Cell-Projection Pumping.

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fibs) and malignant cells (MCs). Others report similar transfer via either tunneling nanotubes (TNTs) or shed membrane vesicles, and this changes the phenotype of recipient cells. Our time-lapse microscopy showed most exchange was from Fibs into MCs, with less in the reverse direction. Although TNTs were seen, we were surprised transfer was not via TNTs but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless by their organellar cargo and the grooves they formed indenting MCs, which was consistent with holotomography. Discrete, rapid, and highly localized transfer events evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell projections normally returns cytoplasm to the cell body. We hypothesize "cell-projection pumping" (CPP), in which cytoplasm in retracting cell projections partially equilibrates into adjacent recipient cells via microfusions that form temporary intercellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modeling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure toward least resistance. Predictions from the model were satisfied when Fibs were cocultured with MCs and fluorescence exchange was related to cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MCs or Fibs was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian intercellular cytoplasmic transfer and communication.

Multimodality labeling strategies for the investigation of nanocrystalline cellulose biodistribution in a mouse model of breast cancer.

METHODS: We have developed a nuclear and fluorescence labeling strategy for nanocrystalline cellulose (CNC), an emerging biomaterial with versatile chemistry and facile preparation from renewable sources. We modified CNC through 1,1'-carbonyldiimidazole (CDI) activation with radiometal chelators desferrioxamine B and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), allowing for the labeling with zirconium-89 (t½â€¯= 78.41 h) and copper-64 (t½â€¯= 12.70 h), respectively, for non-invasive positron emission tomography (PET) imaging. The far-red fluorescent dye Cy5 was added for ex vivo optical imaging, microscopy and flow cytometry. The multimodal CNC were evaluated in the syngeneic orthotopic 4T1 tumor model of human stage IV breast cancer. RESULTS: Modified CNC exhibited low cytotoxicity in RAW 264.7 macrophages over 96 h, and high radiolabel stability in vitro. After systemic administration, radiolabeled CNC were rapidly sequestered to the organs of the reticulo-endothelial system (RES), indicating immune recognition and no passive tumor targeting by the enhanced permeability and retention (EPR) effect. Modification with NOTA was a more favorable strategy in terms of radiolabeling yield, specific radioactivity, and both the radiolabel and dispersion stability in physiological conditions. Flow cytometry analysis of Cy5-positive immune cells from the spleen and tumor corroborated the uptake of CNC to phagocytic cells. CONCLUSIONS: Future studies on the in vivo behavior of CNC should be concentrated on improving the nanomaterial stability and circulation half-life under physiological conditions and optimizing further the labeling yields for the multimodality imaging strategy presented. ADVANCES IN KNOWLEDGE: Our studies constitute one of the first accounts of a multimodality nuclear and fluorescent probe for the evaluation of CNC biodistribution in vivo and outline the pitfalls in radiometal labeling strategies for future evaluation of targeted CNC-based drug delivery systems. IMPLICATIONS FOR PATIENT CARE: Quantitative and sensitive molecular imaging methods provide information on the structure-activity relationships of the nanomaterial and guide the translation from in vitro models to clinically relevant animal models.

Conformation-specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen.

We engineered and employed a chaperone-like amyloid-binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross-reacted with amyloid beta-peptide (Aß42) protofibrils, but not with Aß40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1-hIAPP complex cross-react with Aß42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation-specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation-sensitive and sequence-independent and can target more than one type of protofibril species.

Author Correction: Structural basis for the regulation of inositol trisphosphate receptors by Ca2+ and IP3.

In the version of this article initially published, one of the PDB codes associated with the study was listed in the data availability statement as 6DBC but should have been 6DRC. The error has been corrected in the HTML and PDF versions of the article.

Structural basis for the regulation of inositol trisphosphate receptors by Ca2+ and IP3.

Inositol trisphosphate receptors (IP3Rs) are ubiquitous Ca2+-permeable channels that mediate release of Ca2+ from the endoplasmic reticulum, thereby regulating numerous processes including cell division, cell death, differentiation and fertilization. IP3Rs are jointly activated by inositol trisphosphate (IP3) and their permeant ion, Ca2+. At high concentrations, however, Ca2+ inhibits activity, ensuring precise spatiotemporal control over intracellular Ca2+. Despite extensive characterization of IP3R, the mechanisms through which these molecules control channel gating have remained elusive. Here, we present structures of full-length human type 3 IP3Rs in ligand-bound and ligand-free states. Multiple IP3-bound structures demonstrate that the large cytoplasmic domain provides a platform for propagation of long-range conformational changes to the ion-conduction gate. Structures in the presence of Ca2+ reveal two Ca2+-binding sites that induce the disruption of numerous interactions between subunits, thereby inhibiting IP3R. These structures thus provide a mechanistic basis for beginning to understand the regulation of IP3R.

Cryo-EM and X-ray structures of TRPV4 reveal insight into ion permeation and gating mechanisms.

The transient receptor potential (TRP) channel TRPV4 participates in multiple biological processes, and numerous TRPV4 mutations underlie several distinct and devastating diseases. Here we present the cryo-EM structure of Xenopus tropicalis TRPV4 at 3.8-Å resolution. The ion-conduction pore contains an intracellular gate formed by the inner helices, but lacks any extracellular gate in the selectivity filter, as observed in other TRPV channels. Anomalous X-ray diffraction analyses identify a single ion-binding site in the selectivity filter, thus explaining TRPV4 nonselectivity. Structural comparisons with other TRP channels and distantly related voltage-gated cation channels reveal an unprecedented, unique packing interface between the voltage-sensor-like domain and the pore domain, suggesting distinct gating mechanisms. Moreover, our structure begins to provide mechanistic insights to the large set of pathogenic mutations, offering potential opportunities for drug development.

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation.

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed 'exomeres' (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.

Myelinating glia differentiation is regulated by extracellular matrix elasticity.

The mechanical properties of living tissues have a significant impact on cell differentiation, but remain unexplored in the context of myelin formation and repair. In the PNS, the extracellular matrix (ECM) incorporates a basal lamina significantly denser than the loosely organized CNS matrix. Inhibition of non-muscle myosin II (NMII) enhances central but impairs peripheral myelination and NMII has been implicated in cellular responses to changes in the elasticity of the ECM. To directly evaluate whether mechanotransduction plays a role in glial cell differentiation, we cultured Schwann cells (SC) and oligodendrocytes (OL) on matrices of variable elastic modulus, mimicking either their native environment or conditions found in injured tissue. We found that a rigid, lesion-like matrix inhibited branching and differentiation of OL in NMII-dependent manner. By contrast, SC developed normally in both soft and stiffer matrices. Although SC differentiation was not significantly affected by changes in matrix stiffness alone, we found that expression of Krox-20 was potentiated on rigid matrices at high laminin concentration. These findings are relevant to the design of biomaterials to promote healing and regeneration in both CNS and PNS, via transplantation of glial progenitors or the implantation of tissue scaffolds.

Photoluminescent carbon nanotubes interrogate the permeability of multicellular tumor spheroids.

Nanomaterials have been extensively investigated for cancer drug delivery and imaging applications. Nanoparticles that show promise in two-dimensional cell culture systems often fail in more complex environments, possibly due to the lack of penetration in dense, three-dimensional structures. Multicellular tumor spheroids are an emerging model system to investigate interactions of nanoparticles with 3D in vitro cell culture environments. Using the intrinsic near-infrared emission of semiconducting carbon nanotubes to optically reconstruct their localization within a three-dimensional volume, we resolved the relative permeability of two different multicellular tumor spheroids. Nanotube photoluminescence revealed that nanotubes rapidly internalized into MCF-7 breast cancer cell-derived spheroids, whereas they exhibited little penetration into spheroids derived from SK-136, a cell line that we developed from murine liver cancer. Characterization of the spheroids by electron microscopy and immunohistochemistry revealed large differences in the extracellular matrix and interstitial spacing, which correlated directly with nanotube penetration. This platform portends a new approach to characterize the permeability of living multicellular environments.

A novel cell-stiffness-fingerprinting analysis by scanning atomic force microscopy: comparison of fibroblasts and diverse cancer cell lines.

Differing stimuli affect cell stiffness while cancer metastasis is associated with reduced cell stiffness. Cell stiffness determined by atomic force microscopy has been limited by measurement over nuclei to avoid spurious substratum effects in thin cytoplasmic domains, and we sought to develop a more complete approach including cytoplasmic areas. Ninety µm square fields were recorded from ten separate sites of cultured human dermal fibroblasts (HDF) and three sites each for melanoma (MM39, WM175, and MeIRMu), osteosarcoma (SAOS-2 and U2OS), and ovarian carcinoma (COLO316 and PEO4) cell lines, each site providing 1024 measurements as 32 × 32 square grids. Stiffness recorded below 0.8 µm height was occasionally influenced by substratum, so only stiffness recorded above 0.8 µm was analysed, but all sites were included for height and volume analysis. COLO316 had the lowest cell height and volume, followed by HDF (p < 0.0001) and then PEO4, SAOS-2, MeIRMu, WM175, U2OS, and MM39. HDF were more stiff than all other cells (p < 0.0001), while in descending order of stiffness were PEO4, COLO316, WM175, SAOS-2, U2OS, MM39, and MeIRMu (p < 0.02). Stiffness fingerprints comprised scattergrams of stiffness values plotted against the height at which each stiffness value was recorded and appeared unique for each cell type studied, although in most cases the overall form of fingerprints was similar, with maximum stiffness at low height measurements and a second lower peak occurring at high-height levels. We suggest that our stiffness-fingerprint analytical method provides a more nuanced description than previously reported and will facilitate study of the stiffness response to cell stimulation.