Crystal structure and functional implication of bacterial STING

AbstractMammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense c-di-GMP generated by the CD-NTase (cGAS/DncV-like nucleotidyltransferase) encoded in the same operon and signal suicide commitment as a defense strategy that restricts phage propagation. However, the precise binding mode of c-di-GMP to bacterial STING and the specific recognition mechanism are still elusive. Here, we determine two complex crystal structures of bacterial STING/c-di-GMP, which provide a clear picture of how c-di-GMP is distinguished from other cyclic dinucleotides. The protein-protein interactions further reveal the driving force behind filament formation of bacterial STING. Finally, we group the bacterial STING into two classes based on the conserved motif in β-strand lid, which dictate their ligand specificity and oligomerization mechanism, and propose an evolution-based model that describes the transition from c-di-GMP-dependent signaling in bacteria to 2’3’-cGAMP-dependent signaling in eukaryotes.

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LRRpredictor—A New LRR Motif Detection Method for Irregular Motifs of Plant NLR Proteins Using an Ensemble of Classifiers

Genes ◽

10.3390/genes11030286 ◽

2020 ◽

Vol 11 (3) ◽

pp. 286 ◽

Cited By ~ 3

Author(s):

Eliza C. Martin ◽

Octavina C. A. Sukarta ◽

Laurentiu Spiridon ◽

Laurentiu G. Grigore ◽

Vlad Constantinescu ◽

...

Keyword(s):

Protein Interactions ◽

Structural Data ◽

Innate Immune ◽

Protein Protein Interactions ◽

Ensemble Of Classifiers ◽

Immune Receptor ◽

Motif Detection ◽

Protein Architecture ◽

Recognition Specificity ◽

Leucine Rich Repeats

Leucine-rich-repeats (LRRs) belong to an archaic procaryal protein architecture that is widely involved in protein–protein interactions. In eukaryotes, LRR domains developed into key recognition modules in many innate immune receptor classes. Due to the high sequence variability imposed by recognition specificity, precise repeat delineation is often difficult especially in plant NOD-like Receptors (NLRs) notorious for showing far larger irregularities. To address this problem, we introduce here LRRpredictor, a method based on an ensemble of estimators designed to better identify LRR motifs in general but particularly adapted for handling more irregular LRR environments, thus allowing to compensate for the scarcity of structural data on NLR proteins. The extrapolation capacity tested on a set of annotated LRR domains from six immune receptor classes shows the ability of LRRpredictor to recover all previously defined specific motif consensuses and to extend the LRR motif coverage over annotated LRR domains. This analysis confirms the increased variability of LRR motifs in plant and vertebrate NLRs when compared to extracellular receptors, consistent with previous studies. Hence, LRRpredictor is able to provide novel insights into the diversification of LRR domains and a robust support for structure-informed analyses of LRRs in immune receptor functioning.

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Identification of Novel Inhibitors against Coactivator Associated Arginine Methyltransferase 1 Based on Virtual Screening and Biological Assays

BioMed Research International ◽

10.1155/2016/7086390 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Fei Ye ◽

Weiyao Zhang ◽

Wenchao Lu ◽

Yiqian Xie ◽

Hao Jiang ◽

...

Keyword(s):

Virtual Screening ◽

Small Molecule ◽

Protein Interactions ◽

Binding Mode ◽

Protein Protein Interactions ◽

Arginine Methyltransferase ◽

Docking Analysis ◽

Biological Assays ◽

Biochemical Assays ◽

Methyltransferase 1

Overexpression of coactivator associated arginine methyltransferase 1 (CARM1), a protein arginine N-methyltransferase (PRMT) family enzyme, is associated with various diseases including cancers. Consequently, the development of small-molecule inhibitors targeting PRMTs has significant value for both research and therapeutic purposes. In this study, together with structure-based virtual screening with biochemical assays, two compounds DC_C11 and DC_C66 were identified as novel inhibitors of CARM1. Cellular studies revealed that the two inhibitors are cell membrane permeable and effectively blocked proliferation of cancer cells including HELA, K562, and MCF7. We further predicted the binding mode of these inhibitors through molecular docking analysis, which indicated that the inhibitors competitively occupied the binding site of the substrate and destroyed the protein-protein interactions between CARM1 and its substrates. Overall, this study has shed light on the development of small-molecule CARM1 inhibitors with novel scaffolds.

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Glycoprotein Ibalpha Inhibitor Complex Crystal Structure at High Resolution.

Blood ◽

10.1182/blood.v114.22.774.774 ◽

2009 ◽

Vol 114 (22) ◽

pp. 774-774

Author(s):

P.A Mcewan ◽

Robert K Andrews ◽

Jonas Emsley

Keyword(s):

Crystal Structure ◽

Shear Stress ◽

Protein Interactions ◽

Platelet Adhesion ◽

Peptide Sequence ◽

Leucine Rich Repeat ◽

Protein Protein Interactions ◽

Leucine Rich Repeats ◽

Von Willebrand ◽

Complex Crystal

Abstract Abstract 774 Introduction: The platelet Glycoprotein Ib/V/IX (GpIb/V/IX) complex is considered a major target for anticoagulant therapy. The primary function of the receptor is to mediate platelet adhesion to von Willebrand factor (VWF) bound to damaged sub-endothelium. This represents the first critical step for platelet adhesion under conditions of high fluid shear stress. GpIb/V/IX is implicated in a number of thrombotic pathological processes such as stroke or myocardial infarction and the bleeding disorders Bernard-Soulier syndrome, platelet type von Willebrand disease (Pt-VWD) and thrombotic thrombocytopenic purpura. We have successfully determined the structure of the GpIbalpha N-terminal domain in complex with a potent (sub nM) 11meric peptide inhibitor (OS1) of the interaction with VWF. Methods: We have determined the crystal structure to 1.8Å resolution using molecular replacement. Results. The peptide sequence CTERMALHNLC was readily identifiable bound to GpIbalpha between the extended regulatory (R) loop and the concave surface of the leucine rich repeats. The peptide adopts one and a half turns of an alpha-helix and contacts three subsites (S1, S2 and S3). S1 and S2 reside within the leucine rich repeats and S3 has a unique feature as this subsite involves contact with the regulatory R-loop stabilizing it in a well defined conformation with helical character. This loop alters conformation between an extended beta-hairpin in the VWF-A1 bound structure and a more compact largely disordered structure in the unliganded structure. In this regard, the Pt-VWD mutations of GpIbalpha, G233V and M239V, which reside in the R-loop act by inducing a beta-conformation and thus result in a high affinity form of the receptor. Conclusions: These studies provide a strategy for targeting the GpIbalpha-VWF interaction using small molecules or alpha-helical peptides exploiting the GpIbalpha subsites described here and acting allosterically to stabilise a low affinity conformation of the receptor with an alpha helical R-loop. Ligand mimetic peptide complex crystal structures for the platelet receptors integrin aIIbb3 with RGD, and alpha2beta1 with a collagen peptide have been described and the former are currently in therapeutic use for treatment of thromboemboletic disorders. Targeting the GpIbalpha-VWF interaction may provide anti-thrombotic drugs which affect platelet adhesion under high shear stress without compromising normal processes of platelet adhesion and aggregation which may be required for normal hemostasis to function. Targeting protein-protein interactions is considered one of the great contemporary challenges in drug discovery. The understanding of how the S1S2S3 subsites provide very effective inhibition of a large protein-protein interaction has wide applicability. LRR proteins are an extended family mediating protein-protein interactions involved in a variety of disease processes such as sepsis, asthma, immunodeficiencies, atherosclerosis, alzheimers (leucine rich repeat kinase) and leukaemia (leucine rich repeat phosphatase). The structural fit of the helical curvature of the peptide with the arc of the leucine rich repeats may provide a basis for further development of alpha-helical peptide mimetics targeting other members of the LRR family which utilize the concave face. Disclosures: No relevant conflicts of interest to declare.

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The Viral Macrodomain Counters Host Antiviral ADP-Ribosylation

Viruses ◽

10.3390/v12040384 ◽

2020 ◽

Vol 12 (4) ◽

pp. 384 ◽

Cited By ~ 13

Author(s):

Yousef M. O. Alhammad ◽

Anthony R. Fehr

Keyword(s):

Immune Responses ◽

Protein Interactions ◽

Innate Immune ◽

Innate Immune Responses ◽

Protein Protein Interactions ◽

Granule Formation ◽

Inflammatory Cytokine Production ◽

Adp Ribosylation ◽

Antiviral Responses ◽

Host Defense Response

Macrodomains, enzymes that remove ADP-ribose from proteins, are encoded by several families of RNA viruses and have recently been shown to counter innate immune responses to virus infection. ADP-ribose is covalently attached to target proteins by poly-ADP-ribose polymerases (PARPs), using nicotinamide adenine dinucleotide (NAD+) as a substrate. This modification can have a wide variety of effects on proteins including alteration of enzyme activity, protein–protein interactions, and protein stability. Several PARPs are induced by interferon (IFN) and are known to have antiviral properties, implicating ADP-ribosylation in the host defense response and suggesting that viral macrodomains may counter this response. Recent studies have demonstrated that viral macrodomains do counter the innate immune response by interfering with PARP-mediated antiviral defenses, stress granule formation, and pro-inflammatory cytokine production. Here, we will describe the known functions of the viral macrodomains and review recent literature demonstrating their roles in countering PARP-mediated antiviral responses.

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ADAR1 limits stress granule formation through both translation-dependent and translation-independent mechanisms

Journal of Cell Science ◽

10.1242/jcs.258783 ◽

2021 ◽

Author(s):

Giulia A. Corbet ◽

James M. Burke ◽

Roy Parker

Keyword(s):

Immune Response ◽

Stress Response ◽

Protein Interactions ◽

Binding Activity ◽

Innate Immune ◽

Protein Protein Interactions ◽

Granule Formation ◽

Translation Inhibition ◽

Translation Repression ◽

Dsrna Binding

Stress granules (SGs) are cytoplasmic assemblies of RNA and protein that form when translation is repressed during the integrated stress response (ISR). SGs assemble from the combination of RNA-RNA, RNA-protein, and protein-protein interactions between mRNPs. The protein Adenosine deaminase acting on RNA 1 (ADAR1) recognizes and modifies dsRNAs within cells to prevent an aberrant innate immune response. ADAR1 localizes to SGs, and since RNA-RNA interactions contribute to SG assembly and dsRNA induces SGs, we examined how ADAR1 affects SG formation. First, we demonstrate that ADAR1 depletion triggers SGs by allowing endogenous dsRNA to activate the ISR through PKR activation and translation repression. However, we also show that ADAR1 limits SG formation independently of translation inhibition. ADAR1 repression of SGs is independent of deaminase activity, but dependent on dsRNA-binding activity, suggesting a model where ADAR1 binding limits RNA-RNA and/or RNA-protein interactions necessary for recruitment to SGs. Given that ADAR1 expression is induced during viral infection, these findings have implications for ADAR1's role in the antiviral response.

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A Cascade of Histone Modifications Induces Chromatin Condensation in Mitosis

Science ◽

10.1126/science.1244508 ◽

2014 ◽

Vol 343 (6166) ◽

pp. 77-80 ◽

Cited By ~ 142

Author(s):

Bryan J. Wilkins ◽

Nils A. Rall ◽

Yogesh Ostwal ◽

Tom Kruitwagen ◽

Kyoko Hiragami-Hamada ◽

...

Keyword(s):

Protein Interactions ◽

Driving Force ◽

Chromatin Condensation ◽

Histone H3 ◽

Aurora B ◽

Histone H4 ◽

Protein Protein Interactions ◽

Metaphase Chromosomes ◽

Spatiotemporal Resolution ◽

Acetyl Group

Metaphase chromosomes are visible hallmarks of mitosis, yet our understanding of their structure and of the forces shaping them is rudimentary. Phosphorylation of histone H3 serine 10 (H3 S10) by Aurora B kinase is a signature event of mitosis, but its function in chromatin condensation is unclear. Using genetically encoded ultraviolet light-inducible cross-linkers, we monitored protein-protein interactions with spatiotemporal resolution in living yeast to identify the molecular details of the pathway downstream of H3 S10 phosphorylation. This modification leads to the recruitment of the histone deacetylase Hst2p that subsequently removes an acetyl group from histone H4 lysine 16, freeing the H4 tail to interact with the surface of neighboring nucleosomes and promoting fiber condensation. This cascade of events provides a condensin-independent driving force of chromatin hypercondensation during mitosis.

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Inhibition of β-catenin/B cell lymphoma 9 protein−protein interaction using α-helix–mimicking sulfono-γ-AApeptide inhibitors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819663116 ◽

2019 ◽

Vol 116 (22) ◽

pp. 10757-10762 ◽

Cited By ~ 14

Author(s):

Peng Sang ◽

Min Zhang ◽

Yan Shi ◽

Chunpu Li ◽

Sami Abdulkadir ◽

...

Keyword(s):

B Cell ◽

Protein Interactions ◽

Rational Design ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Binding Mode ◽

Protein Protein Interactions ◽

Protein Protein Interaction ◽

Biological Potential ◽

Α Helix

The rational design of α-helix–mimicking peptidomimetics provides a streamlined approach to discover potent inhibitors for protein−protein interactions (PPIs). However, designing cell-penetrating long peptidomimetic scaffolds equipped with various functional groups necessary for interacting with large protein-binding interfaces remains challenging. This is particularly true for targeting β-catenin/BCL9 PPIs. Here we designed a series of unprecedented helical sulfono-γ-AApeptides that mimic the binding mode of the α-helical HD2 domain of B Cell Lymphoma 9 (BCL9). Our studies show that sulfono-γ-AApeptides can structurally and functionally mimic the α-helical domain of BCL9 and selectively disrupt β-catenin/BCL9 PPIs with even higher potency. More intriguingly, these sulfono-γ-AApeptides can enter cancer cells, bind with β-catenin and disrupt β-catenin/BCL9 PPIs, and exhibit excellent cellular activity, which is much more potent than the BCL9 peptide. Furthermore, our enzymatic stability studies demonstrate the remarkable stability of the helical sulfono-γ-AApeptides, with no degradation in the presence of pronase for 24 h, augmenting their biological potential. This work represents not only an example of helical sulfono-γ-AApeptides that mimic α-helix and disrupt protein–protein interactions, but also an excellent example of potent, selective, and cell-permeable unnatural foldameric peptidomimetics that disrupt the β-catenin/BCL9 PPI. The design of helical sulfono-γ-AApeptides may lead to a new strategy to modulate a myriad of protein–protein interactions.

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Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations

10.1101/435651 ◽

2018 ◽

Author(s):

Ryo Hayama ◽

Mirco Sorci ◽

John J. Keating ◽

Lee M. Hecht ◽

Joel L. Plawsky ◽

...

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Proteins ◽

Binding Mode ◽

Nucleocytoplasmic Transport ◽

Disordered Proteins ◽

Protein Protein Interactions ◽

Force Microscopy ◽

Intrinsically Disordered ◽

Mutant Variant

ABSTRACTProtein-protein interactions are central to biological processes and the methods to thoroughly characterize them are of great interest. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions involved in nucleocytoplasmic transport as a model system, we examined the utility of three surface-based methods in characterizing rapid interactions involving intrinsically disordered proteins: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the existence of previously overlooked mass transport limitations was revealed. Additional experiments with a loss-of-interaction mutant variant demonstrated the existence of additional physical events and an uncharacterized binding mode. These results suggest the binding events that take place on the surface are more complex than initially assumed, prompting a need for re-interpretation of previous data.

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Structural features underlying the activity of benzimidazole derivatives that target phosphopeptide recognition by the tandem BRCT domain of the BRCA1 protein

10.1101/555623 ◽

2019 ◽

Author(s):

Vadiraj Kurdekar ◽

Saranya Giridharan ◽

Jasti Subbarao ◽

Mamatha B. Nijaguna ◽

Jayaprakash Periasamy ◽

...

Keyword(s):

Dna Damage ◽

Protein Interactions ◽

Binding Mode ◽

Structural Features ◽

Benzimidazole Derivatives ◽

Protein Protein Interactions ◽

Brca1 Protein ◽

Cellular Assays ◽

Intracellular Signals ◽

Combine Structure

AbstractThe tandem BRCT (tBRCT) domains of BRCA1 engage pSer-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, a benzimidazole that represents a first selective small molecule inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domains, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features that underlie the biochemical and cellular activities of Bractoppin. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing single residue substitutions that alter K1702, a key residue mediating phosphopeptide recognition (K1702A), or alter hydrophobic residues (F1662R or L1701K) that adjoin the pSer-recognition site. However, mutation of BRCA1 tBRCT residue M1775R, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding. Collectively, these findings confirm a binding mode for Bractoppin that blocks the phosphopeptide-binding site via structural features distinct from the substrate phosphopeptide. We explored these structural features through structure-guided chemical elaboration of Bractoppin, synthesizing analogs bearing modifications on the left and right hand side (LHS/RHS) of Bractoppin’s benzimidazole ring. Characterization of these analogs in biochemical assay reveal structural features underlying potency. Analogs where the LHS phenyl is replaced by cyanomethyl (2091) and 4-methoxyphenoxypropyl (2113) conceptualized from structure-guided strategies like GIST and dimer interface analysis expose the role of phenyl and isopropyl as critical hydrophobic anchors. Two Bractoppin analogs, 2088 and 2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the biochemical and cellular activity of a novel benzimidazole inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target the protein-protein interactions of this previously undrugged family of protein domains.

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Complementation of an Escherichia coli DnaK Defect by Hsc70-DnaK Chimeric Proteins

Journal of Bacteriology ◽

10.1128/jb.186.18.6248-6253.2004 ◽

2004 ◽

Vol 186 (18) ◽

pp. 6248-6253 ◽

Cited By ~ 9

Author(s):

Jean-Philippe Suppini ◽

Mouna Amor ◽

Jean-Hervé Alix ◽

Moncef M. Ladjimi

Keyword(s):

Escherichia Coli ◽

High Temperatures ◽

Protein Interactions ◽

Peptide Binding ◽

Binding Domain ◽

Protein Protein Interactions ◽

Unfolded Proteins ◽

E Coli ◽

Hsp70 Family ◽

Phage Propagation

ABSTRACT Escherichia coli DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein (Hsp70) family that show strong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones. We show here that, while the DnaK protein is, as expected, able to complement an E. coli dnaK mutant strain for growth at high temperatures and λ phage propagation, Hsc70 protein is not. However, an Hsc70 in which the peptide-binding domain has been replaced by that of DnaK is able to complement this strain for both phenotypes, suggesting that the peptide-binding domain of DnaK is essential to fulfill the specific functions of this protein necessary for growth at high temperatures and for λ phage replication. The implications of these findings on the functional specificities of the Hsp70s and the role of protein-protein interactions in the DnaK chaperone system are discussed.

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