C-terminal Binding Site Research Articles

Essential role of N-terminal SAM regions in STIM1 multimerization and function

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

VCIP135 associates with both the N- and C-terminal regions of p97 ATPase

Two distinct p97ATPase-mediated membrane fusion pathways are required for Golgi and endoplasmic reticulum (ER) biogenesis, namely, the p97/p47 pathway and the p97/p37 pathway. p97 (VCP)/p47 complex-interacting protein p135 (VCIP135) is necessary for both of these pathways. Although VCIP135 is known to form a complex with p97 in the cytosol, the role of this complex in Golgi and ER biogenesis has remained unclear. In this study, we demonstrated that VCIP135 has two distinct p97-binding sites at its N- and C-terminal regions. In particular, the C-terminal binding site includes the SHP motif, which is also found in other p97-binding proteins, such as p47, p37, and Ufd1. We also clarified that VCIP135 binds to both the N- and C-terminal regions of p97; that is, the N- and C-terminal binding sites in VCIP135 interact with the C- and N-terminal regions of p97, respectively. These two interactions within the complex are synchronously controlled by the nucleotide state of p97. We next generated VCIP135 mutants lacking each of the p97-binding sites to investigate their functions in living cells and clarified that VCIP135 is involved in Golgi and ER biogenesis through its two distinct interactions with p97. VCIP135 is hence a unique p97-binding protein that functions by interacting with both the N-and C-terminal regions of p97, which strongly suggests that it plays crucial roles in p97-mediated events.

ABRAXAS1 orchestrates BRCA1 activities to counter genome destabilizing repair pathways—lessons from breast cancer patients

It has been well-established that mutations in BRCA1 and BRCA2, compromising functions in DNA double-strand break repair (DSBR), confer hereditary breast and ovarian cancer risk. Importantly, mutations in these genes explain only a minor fraction of the hereditary risk and of the subset of DSBR deficient tumors. Our screening efforts identified two truncating germline mutations in the gene encoding the BRCA1 complex partner ABRAXAS1 in German early-onset breast cancer patients. To unravel the molecular mechanisms triggering carcinogenesis in these carriers of heterozygous mutations, we examined DSBR functions in patient-derived lymphoblastoid cells (LCLs) and in genetically manipulated mammary epithelial cells. By use of these strategies we were able to demonstrate that these truncating ABRAXAS1 mutations exerted dominant effects on BRCA1 functions. Interestingly, we did not observe haploinsufficiency regarding homologous recombination (HR) proficiency (reporter assay, RAD51-foci, PARP-inhibitor sensitivity) in mutation carriers. However, the balance was shifted to use of mutagenic DSBR-pathways. The dominant effect of truncated ABRAXAS1 devoid of the C-terminal BRCA1 binding site can be explained by retention of the N-terminal interaction sites for other BRCA1-A complex partners like RAP80. In this case BRCA1 was channeled from the BRCA1-A to the BRCA1-C complex, which induced single-strand annealing (SSA). Further truncation, additionally deleting the coiled-coil region of ABRAXAS1, unleashed excessive DNA damage responses (DDRs) de-repressing multiple DSBR-pathways including SSA and non-homologous end-joining (NHEJ). Our data reveal de-repression of low-fidelity repair activities as a common feature of cells from patients with heterozygous mutations in genes encoding BRCA1 and its complex partners.

Blockade of Activin Receptor IIB Protects Arthritis Pathogenesis by Non-Amplification of Activin A-ACVR2B-NOX4 Axis Pathway.

Although activin receptor IIB (ACVR2B) is emerging as a novel pathogenic receptor, its ligand and assembled components (or assembly) are totally unknown in the context of osteoarthritis (OA) pathogenesis. The present results suggest that upregulation of ACVR2B and its assembly could affect osteoarthritic cartilage destruction. It is shown that the ACVR2B ligand, activin A, regulates catabolic factor expression through ACVR2B in OA development. Activin A Tg mice (Col2a1-Inhba) exhibit enhanced cartilage destruction, whereas heterozygous activin A KO mice (Inhba+/- ) show protection from cartilage destruction. In silico analysis suggests that the Activin A-ACVR2B axis is involved in Nox4-dependent ROS production. Activin A Tg:Nox4 KO (Col2a1-Inhba:Nox4-/- ) mice show inhibition of experimental OA pathogenesis. NOX4 directly binds to the C-terminal binding site on ACVR2B-ACVR1B and amplifies the pathogenic signal for cartilage destruction through SMAD2/3 signaling. Together, the findings reveal that the ACVR2B assembly, which comprises Activin A, ACVR2B, ACVR1B, Nox4, and AP-1-induced HIF-2α, accelerates OA development. Furthermore, it is shown that shRNA-mediated ACVR2B knockdown or trapping ligands of ACVR2B abrogate OA development by competitively disrupting the ACVR2B-Activin A interaction. These results suggest that the ACVR2B assembly is required to amplify osteoarthritic cartilage destruction and could be a potential therapeutic target in efforts to treat OA.

Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70

Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional prosurvival protein that binds to several intracellular targets and promotes cell survival. HSP70 and Raf-1 are important targets of Bag-1; however, the protective function of Bag-1 in nucleus pulposus (NP) cells remains unclear. In this study, we determined the effects of Bag-1 on NP cells under oxidative stress induced by treatment with hydrogen peroxide (H2O2). We found that Bag-1 was bound to HSP70, but Bag-1-Raf1 binding did not occur in NP cells. Bag-1 overexpression in NP cells enhanced cell viability and mitochondrial function and significantly suppressed p38/MAPKs phosphorylation during oxidative stress, although NP cells treated with a Bag-1 C-terminal inhibitor, which is the binding site of HSP70 and Raf-1, decreased cell viability and mitochondrial function during oxidative stress. Furthermore, the phosphorylation of the ERK/MAPKs was significantly increased in Bag-1 C-terminal inhibitor-treated NP cells without H2O2 treatment but did not change with H2O2 exposure. The phosphorylation of Raf-1 was not influenced by Bag-1 overexpression or Bag-1 C-terminal binding site inhibition. Overall, the results suggest that Bag-1 preferentially interacts with HSP70, rather than Raf-1, to protect NP cells against oxidative stress.

A multipronged footprinting map validates the predicted structure of stress biomarker Neuropeptide Y (NPY) and its binding with novel peptide binders.

Neuropeptide Y (NPY) signals important stress and mood phenotypes in the brain. Peptide biological recognition elements (BREs) based on phage display can be used in hormone biosensors for stress and weight management. We employed protein footprinting mass spectrometry (PF-MS) to probe NPY structure and provide readout of NPY residues responsive to BRE binding. Three complementary PF-MS approaches using hydroxyl radical, trifluoromethyl radical, and carboxyl chemistries were applied independently. This multipronged approach mapped changes in NPY solvent accessibility with 42% labeling coverage.The location and mechanism of binding of two BREs (N2, N3) with NPY was different. Deprotection of NPY residues due to N2 binding suggest an extended NPY conformation, whereas the absence of deprotection in the NPY-N3 complex points to a hairpin conformation. Protection of sites in the NPY-N3 complex agree with reported MD results, confirming a C-terminal binding site. A published NMR structure of NPY shows multiple distinct conformations, including an extended structure with unfolded N-terminus and a poly-proline (PP) α-helix that is back folded onto the NPY C-terminus. AlphaFold2 predicted a PP α-helix structure for NPY. Knowing the true NPY conformation is essential to optimize sensors based upon the NPY-N2 and NPY-N3 complexes. To this end, we reversed the order of PF-MS experiments via a “digest and label” strategy. We found the baseline structure of NPY has a higher order structure, validating the AlphaFold2 prediction. Residue level PF-MS results from this approach are compared to those from footprinting of the intact NPY-BRE complexes to decipher the structural factors impacting NPY solvent accessibility.

The distinct roles of the N-terminal and C-terminal ADP-ribose binding sites in TRPM2 channel

TRPM2 is a calcium-permeable, non-selective cation channel present in different species from unicellular Choanoflagellates to human. TRPM2 plays an important role in the survival of early species and is critically involved in diverse physiological processes including core body temperature regulation, immune response, insulin secretion, and apoptosis. TRPM2 is polymodal and can be activated by a wide range of stimuli including warm temperature, oxidative stress and NAD+-related metabolites such as ADP-ribose (ADPR).

Combination of two monoclonal antibodies with SOCS1 N- and C-terminal binding sites to address SOCS1 status in B cells and B-cell lymphoma.

Accumulating studies show that the tumour suppressor SOCS1 is one of the most frequently mutated genes in lymphomas, often affecting the coding sequence of SOCS1 protein. Depending on the type of mutation and lymphoma concerned, SOCS1mutations have different impacts on progression-free and overall survival. Two antibodies binding the N and C terminals of SOCS1 would be a suitable 'test pair' to identify truncated versions of SOCS1. We, therefore, compared the C-terminal antibody 424C with the N-terminal antibody 4H1. As 424C has already been characterised, we performed a comparative analysis of anti-SOCS1 antibody 4H1 using immunohistochemistry on human tonsil tissue and chamber slides, immunoblots on SOCS1 wildtype and mutated transfected HEK293T cells and lymphoma cell lines and cross-reactivity analysis and epitope mapping with protein microarrays. Compared with 424C, anti-SOCS1 antibody 4H1showed various cross-reactions with other proteins resulting in a 'pancellular' immunohistochemical staining pattern in FFPE lymphoid tissue. Like 424C, 4H1 identified SOCS1 wildtype and SOCS1mutations in immunoblot experiments but also bound an unknown protein with high intensity. Anti-SOCS1 antibody 4H1may be useful in a molecular setting but is disqualified as an immunohistochemical diagnostic tool due to its very broad non-specific binding.

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

Abstract 2450: A systemic approach to decipher the interactome of RET receptor isoforms

Abstract The REarranged during Transfection (RET) receptor tyrosine kinase is pivotal for normal tissue development, but is also an oncogene driver involved in several human cancers. Alternative splicing at the 3' end of the RET gene leads to expression of two conserved protein isoforms, RET9 and RET51, that differ in their subcellular localization and protein trafficking, as well as their functional roles in tumorigenesis and metastatic processes. Importantly, RET9 and RET51 have unique C-terminal phospho-tyrosine binding sites, suggesting that they may also differ in their interactomes. We used a combination of cell-based screening and in silico approaches to identify novel potential interaction partners of RET isoforms. We performed a Mammalian Membrane Two-Hybrid (MaMTH) screen using a library of SH2 domain-containing adaptor and signaling proteins, to identify interactions with each RET isoform. We complemented these studies by using sequence homology detection models (HMM, PSSM), based on SH2 domain sequences known to interact with RET, to rank the SH2 library members and predict novel interactions. Independently, we compared published consensus binding sequences for each SH2 domain library member with predicted RET phosphotyrosine motifs to identify potential interactors. Predicted interactions were validated in co-immunoprecipitation assays. We confirmed previously known interactions of RET with SH2 domain proteins including SHC1, GRB2 and GRB10, and identified additional novel RET-binding proteins, a subset of which showed differential interactions that were mediated through RET isoform-specific docking sites. Our results suggest that combinations of distinct interaction partners may contribute to RET isoform-specific functions. Together, our research has developed a systematic approach to map and characterize RET isoform interactions. Our data suggest that no single method identified all confirmed RET interactions, and that a combination of multiple approaches improves characterization of growth factor receptor interactomes. Citation Format: Samira Kheitan, Annika E. Pedersen, Brandy D. Hyndman, Luka Drecun, Punit Saraon, Igor Stagliar, Lois M. Mulligan. A systemic approach to decipher the interactome of RET receptor isoforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2450.

Unraveling the implications of multiple histidine residues in the potent antimicrobial peptide Gaduscidin-1

The development of antimicrobial peptides (AMPs) as potential therapeutics requires resolving the foundational principles behind their structure-activity relationships. The role of histidine residues within AMPs remains a mystery despite the fact that several potent peptides containing this amino acid are being considered for further clinical development. Gaduscidin-1 (Gad-1) is a potent AMP from Atlantic cod fish that has a total of five His residues. Herein, the role of His residues and metal-potentiated activity of Gad-1 was studied. The five His residues contribute to the broad-spectrum activity of Gad-1. We demonstrated that Gad-1 can coordinate two Cu2+ ions, one at the N-terminus and one at the C-terminus, where the C-terminal binding site is a novel Cu2+ binding motif. High affinity Cu2+ binding at both sites was observed using mass spectrometry and isothermal titration calorimetry. Electron paramagnetic resonance was used to determine the coordination environment of the Cu2+ ions. Cu2+ binding was shown to be responsible for an increase in antimicrobial activity and a new mode of action. Along with the traditional AMP mode of action of pore formation, Gad-1 in the presence of Cu2+ (per)oxidizes lipids. Importantly, His3, His11, His17, and His21 were found to be important to lipid (per)oxidation. This insight will help further understand the inclusion and role of His residues in AMPs, the role of the novel C-terminal binding site, and can contribute to the field of designing potent AMPs that bind metal ions to potentiate activity.

Actin flow-dependent and -independent force transmission through integrins

Integrin-dependent adhesions mediate reciprocal exchange of force and information between the cell and the extracellular matrix. These effects are attributed to the "focal adhesion clutch," in which moving actin filaments transmit force to integrins via dynamic protein interactions. To elucidate these processes, we measured force on talin together with actin flow speed. While force on talin in small lamellipodial adhesions correlated with actin flow, talin tension in large adhesions further from the cell edge was mainly flow-independent. Stiff substrates shifted force transfer toward the flow-independent mechanism. Flow-dependent force transfer required talin's C-terminal actin binding site, ABS3, but not vinculin. Flow-independent force transfer initially required vinculin and at later times the central actin binding site, ABS2. Force transfer through integrins thus occurs not through a continuous clutch but through a series of discrete states mediated by distinct protein interactions, with their ratio modulated by substrate stiffness.

The PP1 regulator PPP1R2 coordinately regulates AURKA and PP1 to control centrosome phosphorylation and maintain central spindle architecture

BackgroundMaintenance of centrosome number in cells is essential for accurate distribution of chromosomes at mitosis and is dependent on both proper centrosome duplication during interphase and their accurate distribution to daughter cells at cytokinesis. Two essential regulators of cell cycle progression are protein phosphatase 1 (PP1) and Aurora A kinase (AURKA), and their activities are each regulated by the PP1 regulatory subunit, protein phosphatase 1 regulatory subunit 2 (PPP1R2). We observed an increase in centrosome number after overexpression of these proteins in cells. Each of these proteins is found on the midbody in telophase and overexpression of PPP1R2 and its mutants increased cell ploidy and disrupted cytokinesis. This suggests that the increase in centrosome number we observed in PPP1R2 overexpressing cells was a consequence of errors in cell division. Furthermore, overexpression of PPP1R2 and its mutants increased midbody length and disrupted midbody architecture. Additionally, we show that overexpression of PPP1R2 alters activity of AURKA and PP1 and their phosphorylation state at the centrosome.ResultsOverexpression of PPP1R2 caused an increase in the frequency of supernumerary centrosomes in cells corresponding to aberrant cytokinesis reflected by increased nuclear content and cellular ploidy. Furthermore, AURKA, PP1, phospho PPP1R2, and PPP1R2 were all localized to the midbody at telophase, and PP1 localization there was dependent on binding of PPP1R2 with PP1 and AURKA as well as its phosphorylation state. Additionally, overexpression of both PPP1R2 and its C-terminal AURKA binding site altered enzymatic activity of AURKA and PP1 at the centrosome and disrupted central spindle structure.ConclusionsResults from our study reveal the involvement of PPP1R2 in coordinating PP1 and AURKA activity during cytokinesis. Overexpression of PPP1R2 or its mutants disrupted the midbody at cytokinesis causing accumulation of centrosomes in cells. PPP1R2 recruited PP1 to the midbody and interference with its targeting resulted in elongated and severely disrupted central spindles supporting an important role for PPP1R2 in cytokinesis.

Synthesis of paramagnetic ligands that target the C-terminal binding site of Hsp90

Identification of a ligand binding site represents the starting point for a structure-based drug development program. Lack of a binding site hampers the development of improved ligands that modulate the protein of interest. In this letter, we describe the development of chemical tools that will allow for elucidation of the Hsp90 C-terminal ligand binding site. Our strategy is based on the preparation of paramagnetic analogs of KU-596, an investigational new drug that is currently undergoing clinical trials for the treatment of neuropathy and interacts with the Hsp90 C-terminal domain. In particular, we report the design and synthesis of three novel paramagnetic analogs of KU-596, which will be used to obtain long range distances for NMR structural studies of Hsp90 in complex with C-terminal ligands.

SAT-313 Synaptojanin 2 Binding Protein Is Required for Efficient Somatostatin Receptor 2A Phosphorylation by G Protein Coupled Receptor Kinases and Signaling to the ERK/MAPK Pathway

Somatostatin receptor 2A (Sst2A) agonists are the primary pharmacological treatment for growth hormone (GH) secreting pituitary tumors to inhibit GH secretion and limit the destructive effects of these tumors. Sst2A agonists are also widely used as imaging agents for neuroendocrine tumors, and are under investigation for treatment of these cancers. However, despite the clinical importance of Sst2A agonists, regulatory mechanisms controlling Sst2A internalization and signaling are not fully understood. Sst2A contains a C-terminal PDZ binding site that serves as a docking platform for PDZ domain containing proteins. In an unbiased screen for PDZ domain proteins that can interact with Sst2A, we identified Synaptojanin 2 binding protein (SYNJ2BP) / Outer mitochondrial protein 25 (OMP25). SYNJ2BP is an outer mitochondrial protein that has been shown previously to promote the internalization of the Activin Type II receptor. We find that SYNJ2BP interacts with Sst2A in a ligand-dependent manner. Importantly, we show that SYNJ2BP promotes interaction of Sst2A with the G protein couple receptor kinase 2 (GRK2), and that siRNA-mediated knockdown of SYNJ2BP dramatically inhibits Sst2A phosphorylation by endogenous GRKs. Moreover, overexpression of SYNJ2BP strongly potentiates Sst2A phosphorylation on the GRK phosphorylation sites. Modulation of SYNJ2BP expression also regulates somatostatin-stimulated β-arrestin recruitment to the plasma membrane. Interestingly, in contrast to the large effects on Sst2A phosphorylation and β-arrestin recruitment, modulation of SYNJ2BP expression only caused a small change in ligand-stimulated receptor internalization. When we assessed downstream signaling, we found that SYNJ2BP overexpression potently stimulated receptor-dependent activation of ERK1/2, but had little effect on the ability of Sst2A to repress forskolin-stimulated cAMP production. Taken together, these data demonstrate for the first time that interaction of Sst2A with a PDZ domain protein affects receptor regulation and signaling. Because multiple PDZ domain containing proteins have been shown to interact with Sst2A, these data also suggest that interaction of Sst2A with this class of proteins may be an important regulatory mechanism to bias its function within the cell.

Phospholipid and Temperature Dependence of TRPV1 Dynamics

The transient receptor potential vanilloid-1 (TRPV1) is a tetrameric nonselective cation channel that acts as a conduit for a variety of external stimuli, including noxious heat, acidosis, and ligand binding, whose signaling ultimately leads to pain sensation. Activation gates the channel, allowing the influx of cations, which subsequently depolarizes the neuron. As an alternative to traditional pain treatments, modulating the behavior of TRPV1 is now the focus of significant drug-discovery efforts. Recently cryo-EM structures of TRPV1 in both the closed(apo) and open states have been reported. Such structures provide an exceptional starting point for structure-based drug discovery, as well as, the unraveling of the biophysical features of temperature activation. Using the crystal structure of the intracellular ankyrin unit and these cryo-EM structures, we have recently built a full length model of the apo-human TRPV1 homo-tetramer. The cryo-EM structure of apo-TRPV1 includes density identified with phospholipid both in the vanilloid binding site and an adjacent region between the S1-S2 helices. The role of these phospholipids in the dynamics and activation of TRPV1 remains elusive. In particular PI(4,5)P2 (phosphatidylinositol 4,5 biphosphate), a known ion channel regulator localized on the inner leaflet of plasma membranes, has been implicated in modulating TRPV1. This work has identified C-terminal binding site residues. To obtain an atomic description of the behavior of the TRPV1 channel with bound phospholipid, we have initiated molecular dynamics simulations in a membrane model (POPC). Simulations include the 1) full apo-TRPV1, 2) channel with a phospholipid bound in the S1-S2 site of each monomer, and 3) inclusion of PI(4,5)P2 in the inner leaflet. These simulations are run at physiological as well as elevated temperatures. The results of these simulations will be presented and discussed with respect to experiment and earlier MD studies. [Support:NIDA-RO1-DA003934 to PHR]

Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes.

RAF family kinases are RAS-activated switches that initiate signaling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1–3. RAF activity is tightly regulated, and inappropriate activation is a frequent cause of cancer4–6. At present, the structural basis for RAF regulation is poorly understood. Here we describe autoinhibited and active state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer, determined using cryo electron microscopy (cryo-EM). A 4.1Å resolution cryo-EM reconstruction reveals an inactive BRAF/MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain (CRD) occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain (RBD) is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding CRD and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, driving formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.

Structure-dependent activation of gene expression by bis-indole and quinoline-derived activators of nuclear receptor 4A2.

Bis-indole derivatives including 1,1-bis(3'-indolyl)-1-(4-chlorophenyl)methane (DIM-C-pPhCl) and substituted quinolines such as chloroquine (CQ) and amodiaquine (AQ) are nuclear receptor 4A2 (NR4A2, Nurr1) ligands, and they exhibit anti-inflammatory activities in mouse and rat models of Parkinson's disease, respectively. However, computational modeling demonstrates that the quinoline derivatives interact with the ligand-binding domain, whereas the bis-indoles preferentially interact with a C-terminal cofactor binding site of NR4A2. In this study, the effects of DIM-C-pPhCl and related analogs were compared with CQ/AQ as inducers of NR4A2-responsive genes including vasoactive intestinal peptide, osteopontin, proopiomelanocortin, and neuropilin 1 in Panc1 and Panc28 pancreatic cancer cells. The results demonstrate that, among the bis-indole analogs, their relative potencies as inducers were structure-gene- and cell context dependent. In contrast, CQ and AQ were significantly less potent than the bis-indole derivatives and, for some of the NR4A2-regulated genes, CQ and AQ were inactive as inducers. These results demonstrate that although bis-indole and quinoline derivatives have been characterized as activators of NR4A2-dependent gene expression, these two classes of compounds exhibit different activities, indicating that they are selective NR4A2 modulators.

STING Polymer Structure Reveals Mechanisms for Activation, Hyperactivation, and Inhibition

How the central innate immune protein, STING, is activated by its ligands remains unknown. Here, using structural biology and biochemistry, we report that the metazoan second messenger 2'3'-cGAMP induces closing of the human STING homodimer and release of the STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 2'3'-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a therapeutic hypothesis for autoimmune syndrome treatment.

Molecular basis of allosteric Orai1 channel activation by STIM1.

Store-operated Ca2+ entry through Orai1 channels is a primary mechanism for Ca2+ entry in many cells and mediates numerous cellular effector functions ranging from gene transcription to exocytosis. Orai1 channels are amongst the most Ca2+ -selective channels known and are activated by direct physical interactions with the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) in response to store depletion triggered by stimulation of a variety of cell surface G-protein coupled and tyrosine kinase receptors. Work in the last decade has revealed that the Orai1 gating process is highly cooperative and strongly allosteric, likely driven by a wave of interdependent conformational changes throughout the protein originating in the peripheral C-terminal ligand binding site and culminating in pore opening. In this review, we survey the structural and molecular features in Orai1 that contribute to channel gating and consider how they give rise to the unique biophysical fingerprint of Orai1 currents.