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.

Structural insights into the HDAC4-MEF2A-DNA complex and its implication in long-range transcriptional regulation.

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7 and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.

Human uridine 5'-monophosphate synthase stores metabolic potential in inactive biomolecular condensates.

Human uridine 5'-monophosphate synthase (HsUMPS) is a bifunctional enzyme that catalyzes the final two steps in de novo pyrimidine biosynthesis. The individual orotate phosphoribosyl transferase and orotidine monophosphate domains have been well characterized, but little is known about the overall structure of the protein and how the organization of domains impacts function. Using a combination of chromatography, electron microscopy, and complementary biophysical methods, we report herein that HsUMPS can be observed in two structurally distinct states, an enzymatically active dimeric form and a nonactive multimeric form. These two states readily interconvert to reach an equilibrium that is sensitive to perturbations of the active site and the presence of substrate. We determined that the smaller molecular weight form of HsUMPS is an S-shaped dimer that can self-assemble into relatively well-ordered globular condensates. Our analysis suggests that the transition between dimer and multimer is driven primarily by oligomerization of the orotate phosphoribosyl transferase domain. While the cellular distribution of HsUMPS is unaffected, quantification by mass spectrometry revealed that de novo pyrimidine biosynthesis is dysregulated when this protein is unable to assemble into inactive condensates. Taken together, our data suggest that HsUMPS self-assembles into biomolecular condensates as a means to store metabolic potential for the regulation of metabolic rates.

Correction: Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

[This corrects the article DOI: 10.1371/journal.pbio.3000901.].

Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRß is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRß signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRß into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1.

Design and optimization of peptide nanoparticles.

BACKGROUND: Various supra-molecular structures form by self-assembly of proteins in a symmetric fashion. Examples of such structures are viruses, some bacterial micro-compartments and eukaryotic vaults. Peptide/protein-based nanoparticles are emerging in synthetic biology for a variety of biomedical applications, mainly as drug targeting and delivery systems or as vaccines. Our self-assembling peptide nanoparticles (SAPNs) are formed by a single peptide chain that consists of two helical coiled-coil segments connected by a short linker region. One helix is forming a pentameric coiled coil while the other is forming a trimeric coiled coil. RESULTS: Here, we were studying in vitro and in silico the effect of the chain length and of point mutations near the linker region between the pentamer and the trimer on the self-assembly of the SAPNs. 60 identical peptide chains co-assemble to form a spherical nanoparticle displaying icosahedral symmetry. We have stepwise reduced the size of the protein chain to a minimal chain length of 36 amino acids. We first used biochemical and biophysical methods on the longer constructs followed by molecular dynamics simulations to study eleven different smaller peptide constructs. We have identified one peptide that shows the most promising mini-nanoparticle model in silico. CONCLUSIONS: An approach of in silico modeling combined with in vitro testing and verification yielded promising peptide designs: at a minimal chain length of only 36 amino acids they were able to self-assemble into proper nanoparticles. This is important since the production cost increases more than linearly with chain length. Also the size of the nanoparticles is significantly smaller than 20 nm, thus reducing the immunogenicity of the particles, which in turn may allow to use the SAPNs as drug delivery systems without the risk of an anaphylactic shock.

Inhibition of the function of class IIa HDACs by blocking their interaction with MEF2.

Enzymes that modify the epigenetic status of cells provide attractive targets for therapy in various diseases. The therapeutic development of epigenetic modulators, however, has been largely limited to direct targeting of catalytic active site conserved across multiple members of an enzyme family, which complicates mechanistic studies and drug development. Class IIa histone deacetylases (HDACs) are a group of epigenetic enzymes that depends on interaction with Myocyte Enhancer Factor-2 (MEF2) for their recruitment to specific genomic loci. Targeting this interaction presents an alternative approach to inhibiting this class of HDACs. We have used structural and functional approaches to identify and characterize a group of small molecules that indirectly target class IIa HDACs by blocking their interaction with MEF2 on DNA.Weused X-ray crystallography and (19)F NMRto show that these compounds directly bind to MEF2. We have also shown that the small molecules blocked the recruitment of class IIa HDACs to MEF2-targeted genes to enhance the expression of those targets. These compounds can be used as tools to study MEF2 and class IIa HDACs in vivo and as leads for drug development.

Rebamipide and Derivatives are Potent, Selective Inhibitors of Histidine Phosphatase Activity of the Suppressor of T Cell Receptor Signaling Proteins.

The suppressor of T cell receptor signaling (Sts) proteins are negative regulators of immune signaling. Genetic inactivation of these proteins leads to significant resistance to infection. From a 590,000 compound high-throughput screen, we identified the 2-(1H)-quinolinone derivative, rebamipide, as a putative inhibitor of Sts phosphatase activity. Rebamipide, and a small library of derivatives, are competitive, selective inhibitors of Sts-1 with IC50 values from low to submicromolar. SAR analysis indicates that the quinolinone, the acid, and the amide moieties are all essential for activity. A crystal structure confirmed the SAR and reveals key interactions between this class of compound and the protein. Although rebamipide has poor cell permeability, we demonstrated that a liposomal preparation can inactivate the phosphatase activity of Sts-1 in cells. These studies demonstrate that Sts-1 enzyme activity can be pharmacologically inactivated and provide foundational tools and insights for the development of immune-enhancing therapies that target the Sts proteins.

Nanomaterials for cancer therapy: current progress and perspectives.

Cancer is a disease with complex pathological process. Current chemotherapy faces problems such as lack of specificity, cytotoxicity, induction of multi-drug resistance and stem-like cells growth. Nanomaterials are materials in the nanorange 1-100 nm which possess unique optical, magnetic, and electrical properties. Nanomaterials used in cancer therapy can be classified into several main categories. Targeting cancer cells, tumor microenvironment, and immune system, these nanomaterials have been modified for a wide range of cancer therapies to overcome toxicity and lack of specificity, enhance drug capacity as well as bioavailability. Although the number of studies has been increasing, the number of approved nano-drugs has not increased much over the years. To better improve clinical translation, further research is needed for targeted drug delivery by nano-carriers to reduce toxicity, enhance permeability and retention effects, and minimize the shielding effect of protein corona. This review summarizes novel nanomaterials fabricated in research and clinical use, discusses current limitations and obstacles that hinder the translation from research to clinical use, and provides suggestions for more efficient adoption of nanomaterials in cancer therapy.

A proposed atomic model of the head-to-tail interaction in the filament structure of vimentin.

Communicated by Ramaswamy H. Sarma.

Estrogen-related receptor alpha directly binds to p53 and cooperatively controls colon cancer growth through the regulation of mitochondrial biogenesis and function.

BACKGROUND: Of the genes that control mitochondrial biogenesis and function, ERRα emerges as a druggable metabolic target to be exploited for cancer therapy. Of the genes mutated in cancer, TP53 remains the most elusive to target. A clear understanding of how mitochondrial druggable targets can be accessed to exploit the underlying mechanism(s) explaining how p53-deficient tumors promote cell survival remains elusive. METHODS: We performed protein-protein interaction studies to demonstrate that ERRα binds to p53. Moreover, we used gene silencing and pharmacological approaches in tandem with quantitative proteomics analysis by SWATH-MS to investigate the role of the ERRα/p53 complex in mitochondrial biogenesis and function in colon cancer. Finally, we designed in vitro and in vivo studies to investigate the possibility of targeting colon cancers that exhibit defects in p53. RESULTS: Here, we are the first to identify a direct protein-protein interaction between the ligand-binding domain (LBD) of ERRα and the C-terminal domain (CTD) of p53. ERRα binds to p53 regardless of p53 mutational status. Furthermore, we show that the ERRα and p53 complex cooperatively control mitochondrial biogenesis and function. Targeting ERRα creates mitochondrial metabolic stresses, such as production of reactive oxygen species (ROS) and mitochondrial membrane permeabilization (MMP), leading to a greater cytotoxic effect that is dependent on the presence of p53. Pharmacological inhibition of ERRα impairs the growth of p53-deficient cells and of p53 mutant patient-derived colon xenografts (PDX). CONCLUSIONS: Therefore, our data suggest that by using the status of the p53 protein as a selection criterion, the ERRα/p53 transcriptional axis can be exploited as a metabolic vulnerability.

Knock-down of the TIM/TIPIN complex promotes apoptosis in melanoma cells.

The Timeless (TIM) and it's interacting partner TIPIN protein complex is well known for its role in replication checkpoints and normal DNA replication processes. Recent studies revealed the involvement of TIM and TIPIN in human malignancies; however, no evidence is available regarding the expression of the TIM/TIPIN protein complex or its potential role in melanoma. Therefore, we investigated the role of this complex in melanoma. To assess the role of the TIM/TIPIN complex in melanoma, we analyzed TIM/TIPIN expression data from the publicly accessible TCGA online database, Western blot analysis, and RT-qPCR in a panel of melanoma cell lines. Lentivirus-mediated TIM/TIPIN knockdown in A375 melanoma cells was used to examine proliferation, colony formation, and apoptosis. A xenograft tumor formation assay was also performed. The TIM/TIPIN complex is frequently overexpressed in melanoma cells compared to normal melanocytes. We also discovered that the overexpression of TIM and TIPIN was significantly associated with poorer prognosis of melanoma patients. Furthermore, we observed that shRNA-mediated knockdown of TIM and TIPIN reduced cell viability and proliferation due to the induction of apoptosis and increased levels of γH2AX, a marker of DNA damage. In a xenograft tumor nude mouse model, shRNA-knockdown of TIM/TIPIN significantly reduced tumor growth. Our results suggest that the TIM/TIPIN complex plays an important role in tumorigenesis of melanoma, which might reveal novel approaches for the development of new melanoma therapies. Our studies also provide a beginning structural basis for understanding the assembly of the TIM/TIPIN complex. Further mechanistic investigations are needed to determine the complex's potential as a biomarker of melanoma susceptibility. Targeting TIM/TIPIN might be a potential therapeutic strategy against melanoma.

Structural Insights into the Induced-fit Inhibition of Fascin by a Small-Molecule Inhibitor.

Tumor metastasis is responsible for ~90% of all cancer deaths. One of the key steps of tumor metastasis is tumor cell migration and invasion. Filopodia are cell surface extensions that are critical for tumor cell migration. Fascin protein is the main actin-bundling protein in filopodia. Small-molecule fascin inhibitors block tumor cell migration, invasion, and metastasis. Here we present the structural basis for the mechanism of action of these small-molecule fascin inhibitors. X-ray crystal structural analysis of a complex of fascin and a fascin inhibitor shows that binding of the fascin inhibitor to the hydrophobic cleft between the domains 1 and 2 of fascin induces a ~35o rotation of domain 1, leading to the distortion of both the actin-binding sites 1 and 2 on fascin. Furthermore, the crystal structures of an inhibitor alone indicate that the conformations of the small-molecule inhibitors are dynamic. Mutations of the inhibitor-interacting residues decrease the sensitivity of fascin to the inhibitors. Our studies provide structural insights into the molecular mechanism of fascin protein function as well as the action of small-molecule fascin inhibitors.

1.6 A crystal structure of the secreted chorismate mutase from Mycobacterium tuberculosis: novel fold topology revealed.

The presence of exported chorismate mutases produced by certain organisms such as Mycobacterium tuberculosis has been shown to correlate with their pathogenicity. As such, these proteins comprise a new group of promising selective drug targets. Here, we report the high-resolution crystal structure of the secreted dimeric chorismate mutase from M. tuberculosis (*MtCM; encoded by Rv1885c), which represents the first 3D-structure of a member of this chorismate mutase family, termed the AroQ(gamma) subclass. Structures are presented both for the unliganded enzyme and for a complex with a transition state analog. The protomer fold resembles the structurally characterized (dimeric) Escherichia coli chorismate mutase domain, but exhibits a new topology, with helix H4 of *MtCM carrying the catalytic site residue missing in the shortened helix H1. Furthermore, the structure of each *MtCM protomer is significantly more compact and only harbors one active site pocket, which is formed entirely by one polypeptide chain. Apart from the structural model, we present evidence as to how the substrate may enter the active site.

Molecular Design of a Minimal Peptide Nanoparticle.

Nanoparticles are getting a great deal of attention in the rapidly developing field of nanomedicine. For example they can be used as drug delivery systems, for imaging applications, or as carriers for synthetic vaccines. Protein-based nanoparticles offer the advantage of biocompatibility and biodegradability thus avoiding some of the major toxicity concerns with nanoparticle associated approaches. Our group has developed self-assembling peptide/protein nanopartices (SAPNs) that are built up from two coiled-coil oligomerization domains joined by a linker region and used them to design subunit vaccines. For drug delivery approaches the SAPNs need to be as small as possible to avoid strong immune responses that could possibly even lead to anaphylaxis. Here we used a computational and biophysical approach to minimize the size of the SAPNs for their use as drug delivery system. We tested different charge distributions on the pentameric and trimeric coiled-coils in silico with molecular dynamics simulations to down-select an optimal design. This design was then investigated in vitro by biophysical methods and we were able to engineer a minimal SAPN of only 11 nm in diameter. Such minimal-sized SAPNs offer new avenues for a safer development as drug delivery systems or other biomedical applications.

Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved.

Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ(gamma) subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5-15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000), Protein Sci. 9, 755-757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 A resolution. The crystals belong to space group P2(1), with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 angstroms, beta = 104.5 degrees. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.

Regulation, Signaling, and Physiological Functions of G-Proteins.

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators.

Homology modelling of the ligand binding domain of mineralocorticoid receptor: close structural kinship with glucocorticoid receptor ligand binding domain and their similar binding mode with DOC (de-oxy corticosterone).

Mineralocorticoids play a major role in regulating sodium and potassium homeostasis and also contribute to the control of blood pressure and in some physiological disorders. The physiological effects of this class of corticosteroids are mediated by ligand-induced nuclear transcription factor, the mineralocorticoid receptor(MR) / glucocorticoid receptor(GR), a member of the steroid / nuclear receptor superfamily. Although the MR interacts with both glucocorticoids and mineralocorticoids, the GR interacts specifically with glucocorticoids. The three dimensional structure of progesterone complexed to its receptor revealed in X-ray diffraction method is utilised to develop a homology model of human mineralocorticoid receptor ligand binding domain (hMR LBD) in a similar fashion as mouse GR LBD was developed previously. The secondary structure of hMR LBD contains eleven helices, eight turns and four sheets. This receptor contains a long helix, H9, with thirty four residues. The 12-residue C-terminal extension (residues 973-984) of hMR LBD, which is essential for hormone binding, is tightly fixed in position by an antiparallel b-sheet interaction. The three dimensional model reveals two polar sites located at the extremities of the elongated hydrophobic ligand-binding pocket (LBP). De-oxy corticosterone (DOC) is docked to the LBs of both hMR LBD and mGR LBD. The difference accessible surface area (DASA) study revealed the interaction zones of both the receptors in complex with DOC. Observations relating to the native and complex proteins revealed a close structural kinship between hMR LBD and mGR LBD.

Nanoscale assemblies and their biomedical applications.

Nanoscale assemblies are a unique class of materials, which can be synthesized from inorganic, polymeric or biological building blocks. The multitude of applications of this class of materials ranges from solar and electrical to uses in food, cosmetics and medicine. In this review, we initially highlight characteristic features of polymeric nanoscale assemblies as well as those built from biological units (lipids, nucleic acids and proteins). We give special consideration to protein nanoassemblies found in nature such as ferritin protein cages, bacterial microcompartments and vaults found in eukaryotic cells and designed protein nanoassemblies, such as peptide nanofibres and peptide nanotubes. Next, we focus on biomedical applications of these nanoscale assemblies, such as cell targeting, drug delivery, bioimaging and vaccine development. In the vaccine development section, we report in more detail the use of virus-like particles and self-assembling polypeptide nanoparticles as new vaccine delivery platforms.

DNA binding by GATA transcription factor suggests mechanisms of DNA looping and long-range gene regulation.

GATA transcription factors regulate transcription during development and differentiation by recognizing distinct GATA sites with a tandem of two conserved zinc fingers, and by mediating long-range DNA looping. However, the molecular basis of these processes is not well understood. Here, we determined three crystal structures of the full DNA-binding domain (DBD) of human GATA3 protein, which contains both zinc fingers, in complex with different DNA sites. In one structure, both zinc fingers wrap around a palindromic GATA site, cooperatively enhancing the binding affinity and kinetic stability. Strikingly, in the other two structures, the two fingers of GATA DBD bind GATA sites on different DNA molecules, thereby bridging two separate DNA fragments. This was confirmed in solution by an in-gel fluorescence resonance energy transfer analysis. These findings not only provide insights into the structure and function of GATA proteins but also shed light on the molecular basis of long-range gene regulation.