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Assignment of electron densities for InsP 3 -binding core domain. A, fitting of 30-Å electron density map of the InsP 3 -binding domain (blue mesh) within the B domains of the cytoplasmic region of InsP 3 R1 tetramer shown in its top view. The x-ray structure of the InsP 3 -binding domain is shown as a ribbon diagram with overlapping electron density map. B, side view of the region extracted from the InsP 3 R1 tetramer as indicated in A with the dashed lines. C, zoom of the region indicated with the dashed lines in A. The white star marks the C terminus of the x-ray structure. The scale bar in A and B is 50 Å and in C is 25 Å.
Source publication
The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified
and formed functional InsP3- and heparin-sensitive channels with a unitary conductance similar to native InsP3Rs. The channel struct...
Citations
... Eventually, the crystal structure of the ligand-binding domain including the suppressor domain and IBCIBC was resolved [ID: 3T8S] at a resolution of 3.0 and 3.8 Å, respectively, in the presence and absence of IP 3 molecule (Lin et al., 2011;Seo et al., 2012). Similarly, different studies reported the 3D structural details of IP 3 R 1 in a closed state at a resolution of >25 Å by using single-particle analysis (da Fonseca et al., 2003;Hamada et al., 2003;Jiang et al., 2002;Serysheva et al., 2003). A consensus of square profile at a lateral dimension of the receptor protein was demonstrated in all studies if viewed from the lumen Serysheva et al., 2003). ...
... Similarly, different studies reported the 3D structural details of IP 3 R 1 in a closed state at a resolution of >25 Å by using single-particle analysis (da Fonseca et al., 2003;Hamada et al., 2003;Jiang et al., 2002;Serysheva et al., 2003). A consensus of square profile at a lateral dimension of the receptor protein was demonstrated in all studies if viewed from the lumen Serysheva et al., 2003). The overall shape and mechanism of conformational changes in IP 3 R were revealed for the first time by these halfway-resolution structures. ...
... In parallel to X-ray crystallographic techniques, the fulllength IP 3 R 1 channel structure was resolved [ID: 3JAV] at a resolution of 4.7 Å by cryo-electron microscopy (cryo-EM) . Using a similar EM technique, Serysheva et al. proposed the structural details of IP 3 R 1 at a better resolution along with the description of backbone topology modeling (Serysheva et al., 2003). Similarly, the full-length IP 3 R 1 channel structure from the rat cerebellum was resolved [ID: 6MU1 and 6MU2] in the apo and bounded state with adenophostin A instead of IP 3 at a resolution of 3.9 and 4.1 Å, respectively (Fan et al., 2018). ...
Calcium signaling has been identified as an important phenomenon in a plethora of cellular processes. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ER-residing intracellular calcium (Ca²⁺) release channels responsible for cell bioenergetics by transferring calcium from the ER to the mitochondria. The recent availability of full-length IP3R channel structure has enabled the researchers to design the IP3 competitive ligands and reveal the channel gating mechanism by elucidating the conformational changes induced by ligands. However, limited knowledge is available for IP3R antagonists and the exact mechanism of action of these antagonists within a tumorigenic environment of a cell. Here in this review a summarized information about the role of IP3R in cell proliferation and apoptosis has been discussed. Moreover, structure and gating mechanism of IP3R in the presence of antagonists have been provided in this review. Additionally, compelling information about ligand-based studies (both agonists and antagonists) has been discussed. The shortcomings of these studies and the challenges toward the design of potent IP3R modulators have also been provided in this review. However, the conformational changes induced by antagonists for channel gating mechanism still display some major drawbacks that need to be addressed. However, the design, synthesis and availability of isoform-specific antagonists is a rather challenging one due to intra-structural similarity within the binding domain of each isoform.
• Highlights
• The intricate complexity of IP3R’s in cellular processes declares them an important target whereby, the recently solved structure depicts the receptor’s potential involvement in a complex network of processes spanning from cell proliferation to cell death.
• Pharmacological inhibition of IP3R attenuates the proliferation or invasiveness of cancers, thus inducing necrotic cell death.
• Despite significant advancements, there is a tremendous need to design new potential hits to target IP3R, based upon 3D structural features and pharmacophoric patterns.
Communicated by Ramaswamy H. Sarma
... These include an IP 3 -binding core (IBC) region and a suppressor domain (residues 600) at the N terminus of the protein. The cytosolic domain also includes a central modulatory region (which mostly interacts with regulatory proteins) and a channel (pore) with six putative transmembrane (TM) domains (residues 2276-2589) near the protein's C terminus [23,[27][28][29]. Recent structural investigations of IP 3 Rs [26,30] and availability of the 3D structure of IP 3 R 3 in apostate and ligand-bound states [30,31] paved the way to study the binding hypothesis of the IP 3 molecule and antagonists to elucidate the effect of IP 3 R inhibition upon channel gating. ...
Inositol 1, 4, 5-trisphosphate receptor (IP3R)-mediated Ca2+ signaling plays a pivotal role in different cellular processes, including cell proliferation and cell death. Remodeling Ca2+ signals by targeting the downstream effectors is considered an important hallmark in cancer progression. Despite recent structural analyses, no binding hypothesis for antagonists within the IP3-binding core (IBC) has been proposed yet. Therefore, to elucidate the 3D structural features of IP3R modulators, we used combined pharmacoinformatic approaches, including ligand-based pharmacophore models and grid-independent molecular descriptor (GRIND)-based models. Our pharmacophore model illuminates the existence of two hydrogen-bond acceptors (2.62 Å and 4.79 Å) and two hydrogen-bond donors (5.56 Å and 7.68 Å), respectively, from a hydrophobic group within the chemical scaffold, which may enhance the liability (IC50) of a compound for IP3R inhibition. Moreover, our GRIND model (PLS: Q2 = 0.70 and R2 = 0.72) further strengthens the identified pharmacophore features of IP3R modulators by probing the presence of complementary hydrogen-bond donor and hydrogen-bond acceptor hotspots at a distance of 7.6–8.0 Å and 6.8–7.2 Å, respectively, from a hydrophobic hotspot at the virtual receptor site (VRS). The identified 3D structural features of IP3R modulators were used to screen (virtual screening) 735,735 compounds from the ChemBridge database, 265,242 compounds from the National Cancer Institute (NCI) database, and 885 natural compounds from the ZINC database. After the application of filters, four compounds from ChemBridge, one compound from ZINC, and three compounds from NCI were shortlisted as potential hits (antagonists) against IP3R. The identified hits could further assist in the design and optimization of lead structures for the targeting and remodeling of Ca2+ signals in cancer.
... Early EM studies on tetrameric IP 3 R structure were performed by negative staining of purified IP 3 R (81, 105-107), and cryo-EM studies were published from several groups (82,108,109); however, their published maps showed different structures, and the discrepancies have been discussed in review papers (110,111). Thereafter, the IP 3 receptor structure at a 17-Å resolution was published, followed by the recent revolution of cryo-EM technology that uncovered the structure of IP 3 R at a 3-to 5-Å resolution (83)(84)(85). ...
In the body, extracellular stimuli produce inositol 1,4,5-trisphosphate (IP 3 ), an intracellular chemical signal that binds to the IP 3 receptor (IP 3 R) to release calcium ions (Ca ²⁺ ) from the endoplasmic reticulum. In the past 40 years, the wide-ranging functions mediated by IP 3 R and its genetic defects causing a variety of disorders have been unveiled. Recent cryo-electron microscopy and X-ray crystallography have resolved IP 3 R structures and begun to integrate with concurrent functional studies, which can explicate IP 3 -dependent opening of Ca ²⁺ -conducting gates placed ∼90 Å away from IP 3 -binding sites and its regulation by Ca ²⁺ . This review highlights recent research progress on the IP 3 R structure and function. We also propose how protein plasticity within IP 3 R, which involves allosteric gating and assembly transformations accompanied by rapid and chronic structural changes, would enable it to regulate diverse functions at cellular microdomains in pathophysiological states.
Expected final online publication date for the Annual Review of Physiology, Volume 82 is February 10, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Structural investigation of IP 3 Rs was pioneered by using the IP 3 R-1 obtained from native tissues, due to its abundant expression in the cerebellum and well established purification strategy (18)(19)(20)(21)(22)(23)(24)(25)(26). Recently, cryo-EM structures of recombinant IP 3 R-3 have been reported and the global architectures of both IP 3 R-1 and IP 3 R-3 share common features (27). ...
Calcium-mediated signaling through inositol 1,4,5-triphosphate receptors (IP 3 Rs) is essential for the regulation of numerous physiological processes, including fertilization, muscle contraction, apoptosis, secretion, and synaptic plasticity. Deregulation of IP 3 Rs leads to pathological calcium signaling and is implicated in many common diseases, including cancer and neurodegenerative, autoimmune, and metabolic diseases. Revealing the mechanism of activation and inhibition of this ion channel will be critical to an improved understanding of the biological processes that are controlled by IP3Rs. Here, we report structural findings of the human type-3 IP 3 R (IP 3 R-3) obtained by cryo-EM (at an overall resolution of 3.8 Å), revealing an unanticipated regulatory mechanism where a loop distantly located in the primary sequence occupies the IP 3 -binding site and competitively inhibits IP 3 binding. We propose that this inhibitory mechanism must differ qualitatively among IP 3 R subtypes because of their diverse loop sequences, potentially serving as a key molecular determinant of subtype-specific calcium signaling in IP 3 Rs. In summary, our structural characterization of human IP 3 R-3 provides critical insights into the mechanistic function of IP3Rs and into subtype-specific regulation of these important calcium-regulatory channels.
... For multicomponent biological complexes, the compositional and conformational heterogeneity is foreseeably inevitable, and will introduce structural discrepancies when different procedures were used to prepare the same complexes. For example, structural studies of the inositol 1,4,5-trisphosphate receptor (IP 3 R) have generated contrastingly different 3D structures by four different research groups ( Jiang et al. 2002;da Fonseca et al. 2003;Serysheva et al. 2003;Sato et al. 2004;Serysheva and Ludtke 2010;Ludtke et al. 2011). Two research groups reported very different structures when they varied the preparation of the protein from different sources using different protocols (da Fonseca et al. 2003;Serysheva et al. 2003;Wolfram et al. 2010;Ludtke et al. 2011). ...
... For example, structural studies of the inositol 1,4,5-trisphosphate receptor (IP 3 R) have generated contrastingly different 3D structures by four different research groups ( Jiang et al. 2002;da Fonseca et al. 2003;Serysheva et al. 2003;Sato et al. 2004;Serysheva and Ludtke 2010;Ludtke et al. 2011). Two research groups reported very different structures when they varied the preparation of the protein from different sources using different protocols (da Fonseca et al. 2003;Serysheva et al. 2003;Wolfram et al. 2010;Ludtke et al. 2011). It is therefore worth reminding the readers that special attention should be paid to the preparation of biological specimens before loading them to EM grids. ...
Abstract was not published with the book. The chapter was prepared in 2012-2013. It described stepwise from sample preparation to data collection and data analysis. Different types of specimens were discussed and the prediction of fast progress was made as well.
... Structural information is also essential for the understanding of ion channel function, despite the fact that it is usually attained with much more difficulty, and by independent means [5]. Cryo-electron microscopy (EM) [6][7][8] and X-ray crystallography [9,10] provide highly comprehensive, but static pictures of channel structures. Single-particle cryo-EM is particularly useful because it provides structural information of ion channels in their native aqueous environment [11,12]. ...
Ion channels are transmembrane proteins that mediate ion transport across biological membranes. Ion channel function is traditionally characterized by electrical parameters acquired with techniques such as patch-clamping and reconstitution in lipid bilayer membranes (BLM) that provide relevant information such as ionic conductance, selectivity, and gating properties. High resolution structural information of ion channels however, requires independent technologies, of which atomic force microscopy (AFM) is the only one that provides topological features of single functional channel proteins in their native environments. To date practically no data exist on direct correlations between electrical features and topological parameters from functional single channel complexes. Here, we report the design and construction of a BLM reconstitution microchamber that supports the simultaneous recording of electrical currents and AFM imaging from single channel complexes. As proof-of-principle, we tested the technique on polycystin-2 (PC2, TRPP2), a TRP channel family member from which we had previously elucidated its tetrameric topology by AFM imaging, and single channel currents by the BLM technique. The experimental setup provided direct structural-functional correlates from PC2 single channel complexes that disclosed novel topological changes between the closed and open sub-conductance states of the functional channel, namely, an inverse correlation between conductance and height of the channel. Unexpectedly, we also disclosed intrinsic PC2 mechanosensitivity in response to external forces. The platform provides a suitable means of accessing topological information to correlate with ion channel electrical parameters essential to understand the physiology of these transmembrane proteins.
... Yoshinori Fujiyoshi, and gave a talk entitled "Structure and Function of IP 3 receptors." Among the audience there were three scientists who later published cryo-EM structures of IP 3 R, Dr. Jiang (Jiang et al., 2002), Dr. Serysheva (Serysheva et al., 2003), and Dr. Sato (Sato et al., 2004). The conference was highly active because it was attended by Professor Joachim Frank (a nobel prize laureate in 2017), a famous pioneer of single particle analysis of EM, but also highly motivated young scientists who were working on cryo-EM or X-ray crystallography of ion channels. ...
... These new aspects were not reported in the papers describing various 3D structures (da Fonseca et al., 2003;Jiang et al., 2002;Serysheva et al., 2003), but we had become strongly confident that these shapes truly represent the structure of IP 3 R based on the data that IP 3 R changes in structure in a manner dependent on calcium ions based on deposit data accumulated in our laboratory. Furthermore, taken together with heparin-gold and limited digestion experiments, we proposed the first model that detailed a long distance from IP 3 -binding site to the channel (Hamada et al., 2003). ...
... Furthermore, taken together with heparin-gold and limited digestion experiments, we proposed the first model that detailed a long distance from IP 3 -binding site to the channel (Hamada et al., 2003). Although Dr. Serysheva had already published a cryo-EM structure (Serysheva et al., 2003) that differed from our mushroom-shaped structure. Subsequently, using modified purification methods this group later published mushroom-like images by cryo-EM (Ludtke et al., 2011). ...
... The gating mechanism of the IP 3 R1 channel by IP 3 , which is produced from phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by being cleaved by phospholipase C (PLC), is an exciting topic, and many groups including ours have shown structural models of IP 3 R1 [15][16][17][18][19][20][21][22]. Especially, Fan et al. demonstrated the cryo-EM (electron microscopy) structure of the tetrameric IP 3 R1 in its apo-state at near atomic resolution level and suggested a critical physical interaction between the cytosolic COOH termini and the amino-terminal domains of adjacent subunits for channel gating [19] (Fig. 1B). ...
Spinocerebellar ataxia (SCA) is a neural disorder, which is caused by degenerative changes in the cerebellum. SCA is primarily characterized by gait ataxia, and additional clinical features include nystagmus, dysarthria, tremors and cerebellar atrophy. Forty-four hereditary SCAs have been identified to date, along with >35 SCA-associated genes. Despite the great diversity and distinct functionalities of the SCA-related genes, accumulating evidence supports the occurrence of a common pathophysiological event among several hereditary SCAs. Altered calcium (Ca2+) homeostasis in the Purkinje cells (PCs) of the cerebellum has been proposed as a possible pathological SCA trigger. In support of this, signaling events that are initiated from or lead to aberrant Ca2+ release from the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), which is highly expressed in cerebellar PCs, seem to be closely associated with the pathogenesis of several SCA types. In this review, we summarize the current research on pathological hereditary SCA events, which involve altered Ca2+ homeostasis in PCs, through IP3R1 signaling.
... Mononuclear blood cells (2 × 10 5 /200 μl) from patient 1 (sample obtained prior to tumour therapy) and a healthy control donor were cultured in triplicate in U-bottom microtitre wells (TPP Techno Plastic Products AG, Trasadingen, Switzerland) in RPMI 1640 medium supplemented with 10 % heat-inactivated AB human serum (Sigma Chemie), 2 mM L-glutamine and 100 U/ml penicillin-streptomycin and incubated with either purified native rat ITPR1 (10 μg/ml) [17], purified human glial fibrillary acidic protein (GFAP) (10 μg/ml) (Merck Chemicals, Darmstadt, Germany) or phosphate buffered saline (PBS) (Euroimmun). The proliferative response was measured by the uptake of 3 H-thymidine (0.5 μCi/well) added 16 h before harvesting on day 3 of culture. ...
Background
Recently, we described a novel autoantibody, anti-Sj/ITPR1-IgG, that targets the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) in patients with cerebellar ataxia. However, ITPR1 is expressed not only by Purkinje cells but also in the anterior horn of the spinal cord, in the substantia gelatinosa and in the motor, sensory (including the dorsal root ganglia) and autonomic peripheral nervous system, suggesting that the clinical spectrum associated with autoimmunity to ITPR1 may be broader than initially thought. Here we report on serum autoantibodies to ITPR1 (up to 1:15,000) in three patients with (radiculo)polyneuropathy, which in two cases was associated with cancer (ITPR1-expressing adenocarcinoma of the lung, multiple myeloma), suggesting a paraneoplastic aetiology. Methods
Serological and other immunological studies, and retrospective analysis of patient records. ResultsThe clinical findings comprised motor, sensory (including severe pain) and autonomic symptoms. While one patient presented with subacute symptoms mimicking Guillain-Barré syndrome (GBS), the symptoms progressed slowly in two other patients. Electrophysiology revealed delayed F waves; a decrease in motor and sensory action potentials and conduction velocities; delayed motor latencies; signs of denervation, indicating sensorimotor radiculopolyneuropathy of the mixed type; and no conduction blocks. ITPR1-IgG belonged to the complement-activating IgG1 subclass in the severely affected patient but exclusively to the IgG2 subclass in the two more mildly affected patients. Cerebrospinal fluid ITPR1-IgG was found to be of predominantly extrathecal origin. A 3H-thymidine-based proliferation assay confirmed the presence of ITPR1-reactive lymphocytes among peripheral blood mononuclear cells (PBMCs). Immunophenotypic profiling of PBMCs protein demonstrated predominant proliferation of B cells, CD4 T cells and CD8 memory T cells following stimulation with purified ITPR1 protein. Patient ITPR1-IgG bound both to peripheral nervous tissue and to lung tumour tissue. A nerve biopsy showed lymphocyte infiltration (including cytotoxic CD8 cells), oedema, marked axonal loss and myelin-positive macrophages, indicating florid inflammation. ITPR1-IgG serum titres declined following tumour removal, paralleled by clinical stabilization. Conclusions
Our findings expand the spectrum of clinical syndromes associated with ITPR1-IgG and suggest that autoimmunity to ITPR1 may underlie peripheral nervous system diseases (including GBS) in some patients and may be of paraneoplastic origin in a subset of cases.
... Cryo-EM and single-particle analysis of detergent-solubilized particles have proven to be the only feasible way to provide structural information of the complete calcium release channel IP3R. Several early attempts to reconstruct IP3R gave inconsistent results (Serysheva et al., 2003;Jiang et al., 2002;Sato et al., 2004). The correct 3D reconstruction emerged after acquisition of better images and rigorous validation of the 3D reconstruction (Ludtke et al., 2011;Ludtke and Serysheva, 2013). ...
This review describes some of the methods for atomic structure refinement (fitting) against medium/high-resolution single-particle cryo-EM reconstructed maps. Some of the tools developed for macromolecular X-ray crystal structure analysis, especially those encapsulating prior chemical and structural information can be transferred directly for fitting into cryo-EM maps. However, despite the similarities, there are significant differences between data produced by these two techniques; therefore, different likelihood functions linking the data and model must be used in cryo-EM and crystallographic refinement. Although tools described in this review are mostly designed for medium/high-resolution maps, if maps have sufficiently good quality, then these tools can also be used at moderately low resolution, as shown in one example. In addition, the use of several popular crystallographic methods is strongly discouraged in cryo-EM refinement, such as 2Fo − Fc maps, solvent flattening, and feature-enhanced maps (FEMs) for visualization and model (re)building. Two problems in the cryo-EM field are overclaiming resolution and severe map oversharpening. Both of these should be avoided; if data of higher resolution than the signal are used, then overfitting of model parameters into the noise is unavoidable, and if maps are oversharpened, then at least parts of the maps might become very noisy and ultimately uninterpretable. Both of these may result in suboptimal and even misleading atomic models.
