Inherent Protein Structural Flexibility at the RNA-binding Interface of L30e

The La and the La-related protein (LARP) superfamily is a diverse class of RNA binding proteins involved in RNA processing, folding, and function. Larp7 binds to the abundant long noncoding 7SK RNA and is required for 7SK ribonucleoprotein (RNP) assembly and function. The 7SK RNP sequesters a pool of the positive transcription elongation factor b (P-TEFb) in an inactive state; on release, P-TEFb phosphorylates RNA Polymerase II to stimulate transcription elongation. Despite its essential role in transcription, limited structural information is available for the 7SK RNP, particularly for protein–RNA interactions. Larp7 contains an N-terminal La module that binds UUU-3′OH and a C-terminal atypical RNA recognition motif (xRRM) required for specific binding to 7SK and P-TEFb assembly. Deletion of the xRRM is linked to gastric cancer in humans. We report the 2.2-Å X-ray crystal structure of the human La-related protein group 7 (hLarp7) xRRM bound to the 7SK stem-loop 4, revealing a unique binding interface. Contributions of observed interactions to binding affinity were investigated by mutagenesis and isothermal titration calorimetry. NMR 13C spin relaxation data and comparison of free xRRM, RNA, and xRRM–RNA structures show that the xRRM is preordered to bind a flexible loop 4. Combining structures of the hLarp7 La module and the xRRM–7SK complex presented here, we propose a structural model for Larp7 binding to the 7SK 3′ end and mechanism for 7SK RNP assembly. This work provides insight into how this domain contributes to 7SK recognition and assembly of the core 7SK RNP.

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Asterix/Gtsf1 links tRNAs and piRNA silencing of retrotransposons

10.1101/2020.08.11.246769 ◽

2020 ◽

Author(s):

Jonathan J. Ipsaro ◽

Paul A. O’Brien ◽

Shibani Bhattacharya ◽

Arthur G. Palmer ◽

Leemor Joshua-Tor

Keyword(s):

Nmr Spectroscopy ◽

Zinc Finger ◽

Biochemical Analysis ◽

Rna Binding ◽

Ltr Retrotransposon ◽

Ltr Retrotransposons ◽

Genomic Integrity ◽

Binding Interface ◽

Pirna Pathway

AbstractThe piRNA pathway safeguards genomic integrity by silencing transposable elements in the germline. While Piwi is the central piRNA factor, others including Asterix/Gtsf1 have also been demonstrated to be critical for effective silencing. Here, using eCLIP with a custom informatic pipeline, we show that Asterix/Gtsf1 specifically binds tRNAs in cellular contexts. We determined the structure of mouse Gtsf1 by NMR spectroscopy and identified the RNA binding interface on the protein’s first zinc finger, which was corroborated by biochemical analysis as well as cryo-EM structures of Gtsf1 in complex with co-purifying tRNA. We further show that LTR retrotransposons are preferentially de-repressed in Asterix mutants. Given the role of tRNAs as LTR retrotransposon primers, our work implicates Asterix/Gtsf1 as exploiting tRNA dependence to identify transposon transcripts and promote piRNA silencing.

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Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain

10.1101/2020.11.30.404905 ◽

2020 ◽

Author(s):

Chao Wu ◽

Abraham J. Qavi ◽

Asmaa Hachim ◽

Niloufar Kavian ◽

Aidan R. Cole ◽

...

Keyword(s):

Rna Binding ◽

Diagnostic Value ◽

Biochemical Properties ◽

Spherical Droplet ◽

N Protein ◽

Exchange Mass Spectrometry ◽

Domain Specific ◽

Terminal Domain ◽

Binding Interface ◽

Hydrogen Deuterium

SummaryNucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.

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UHM–ULM interactions in the RBM39–U2AF65 splicing-factor complex

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316001248 ◽

2016 ◽

Vol 72 (4) ◽

pp. 497-511 ◽

Cited By ~ 17

Author(s):

Galina A. Stepanyuk ◽

Pedro Serrano ◽

Eigen Peralta ◽

Carol L. Farr ◽

Herbert L. Axelrod ◽

...

Keyword(s):

Protein Interactions ◽

Rna Binding ◽

Splicing Factor ◽

Structural Basis ◽

Titration Calorimetry ◽

Protein Protein Interactions ◽

Rrm Domain ◽

Nmr Structures ◽

Binding Interface ◽

Cell Extracts

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein–protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39–U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35–U2AF65 and RBM39–SF3b155, the RBM39-UHM–U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM–ULM binding interface, providing a rationale for the known specificity of UHM–ULM interactions. This study therefore establishes a structural basis for specific UHM–ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.

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Simulation of Molecular Dynamics of SARS-CoV-2 S-Protein in the Presence of Multiple Arbidol Molecules: Interactions and Binding Mode Insights

Viruses ◽

10.3390/v14010119 ◽

2022 ◽

Vol 14 (1) ◽

pp. 119

Author(s):

Sophia S. Borisevich ◽

Edward M. Khamitov ◽

Maxim A. Gureev ◽

Olga I. Yarovaya ◽

Nadezhda B. Rudometova ◽

...

Keyword(s):

Molecular Dynamics ◽

Antiviral Activity ◽

Viral Entry ◽

Binding Sites ◽

Surface Protein ◽

Protein S ◽

Binding Mode ◽

Structural Flexibility ◽

Binding Interface ◽

Dynamics Simulations

In this work, we evaluated the antiviral activity of Arbidol (Umifenovir) against SARS-CoV-2 using a pseudoviral system with the glycoprotein S of the SARS-CoV-2 virus on its surface. In order to search for binding sites to protein S of the virus, we described alternative binding sites of Arbidol in RBD and in the ACE-2-RBD complex. As a result of our molecular dynamics simulations combined with molecular docking data, we note the following fact: wherever the molecules of Arbidol bind, the interaction of the latter affects the structural flexibility of the protein. This interaction may result both in a change in the shape of the domain–enzyme binding interface and simply in a change in the structural flexibility of the domain, which can subsequently affect its affinity to the enzyme. In addition, we examined the possibility of Arbidol binding in the stem part of the surface protein. The possibility of Arbidol binding in different parts of the protein is not excluded. This may explain the antiviral activity of Arbidol. Our results could be useful for researchers searching for effective SARS-CoV-2 virus inhibitors targeting the viral entry stage.

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Structural analyses of the CRISPR protein Csc2 reveal the RNA-binding interface of the type I-D Cas7 family

RNA Biology ◽

10.4161/rna.29893 ◽

2014 ◽

Vol 11 (8) ◽

pp. 1072-1082 ◽

Cited By ~ 11

Author(s):

Ajla Hrle ◽

Lisa-Katharina Maier ◽

Kundan Sharma ◽

Judith Ebert ◽

Claire Basquin ◽

...

Keyword(s):

Rna Binding ◽

Type I ◽

Binding Interface

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Structural basis for the recognition of transiently structured AU-rich elements by Roquin

Nucleic Acids Research ◽

10.1093/nar/gkaa465 ◽

2020 ◽

Author(s):

Oliver Binas ◽

Jan-Niklas Tants ◽

Stephen A Peter ◽

Robert Janowski ◽

Elena Davydova ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Regulatory Elements ◽

Structural Basis ◽

Mrna Regulation ◽

Short Linear Motifs ◽

Binding Interface ◽

Multiple Copies ◽

Linear Motifs ◽

Fine Tune

Abstract Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3′-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3′-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.

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Molecular Dynamics Simulations of Influenza A Virus NS1 Reveal a Remarkably Stable RNA-Binding Domain Harboring Promising Druggable Pockets

Viruses ◽

10.3390/v12050537 ◽

2020 ◽

Vol 12 (5) ◽

pp. 537

Author(s):

Hiba Abi Hussein ◽

Colette Geneix ◽

Camille Cauvin ◽

Daniel Marc ◽

Delphine Flatters ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Influenza A ◽

Rna Binding ◽

Biological Activities ◽

Binding Domain ◽

Structural Protein ◽

Binding Interface ◽

Rna Binding Domain ◽

Dynamics Simulations

The non-structural protein NS1 of influenza A viruses is considered to be the major antagonist of the interferon system and antiviral defenses of the cell. It could therefore represent a suitable target for novel antiviral strategies. As a first step towards the identification of small compounds targeting NS1, we here investigated the druggable potential of its RNA-binding domain since this domain is essential to the biological activities of NS1. We explored the flexibility of the full-length protein by running molecular dynamics simulations on one of its published crystal structures. While the RNA-binding domain structure was remarkably stable along the simulations, we identified a flexible site at the two extremities of the “groove” that is delimited by the antiparallel α-helices that make up its RNA-binding interface. This groove region is able to form potential binding pockets, which, in 60% of the conformations, meet the druggability criteria. We characterized these pockets and identified the residues that contribute to their druggability. All the residues involved in the druggable pockets are essential at the same time to the stability of the RNA-binding domain and to the biological activities of NS1. They are also strictly conserved across the large sequence diversity of NS1, emphasizing the robustness of this search towards the identification of broadly active NS1-targeting compounds.

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Discovery of a cryptic allosteric site in Ebola’s ‘undruggable’ VP35 protein using simulations and experiments

10.1101/2020.02.09.940510 ◽

2020 ◽

Cited By ~ 1

Author(s):

Matthew A. Cruz ◽

Thomas E. Frederick ◽

Sukrit Singh ◽

Neha Vithani ◽

Maxwell I. Zimmerman ◽

...

Keyword(s):

Innate Immunity ◽

Adaptive Sampling ◽

Ebola Virus ◽

Rna Binding ◽

Experimental Tests ◽

Allosteric Site ◽

Detection Algorithms ◽

Binding Interface ◽

Protein Nucleic Acid ◽

Nucleic Acid Interactions

AbstractMany proteins are classified as ‘undruggable,’ especially those that engage in protein-protein and protein-nucleic acid interactions. Discovering ‘cryptic’ pockets that are absent in available structures but open due to protein dynamics could provide new druggable sites. Here, we integrate atomically-detailed simulations and biophysical experiments to search for cryptic pockets in viral protein 35 (VP35) from the highly lethal Ebola virus. VP35 plays multiple essential roles in Ebola’s replication cycle, including binding the viral RNA genome to block a host’s innate immunity. However, VP35 has so far proved undruggable. Using adaptive sampling simulations and allosteric network detection algorithms, we uncover a cryptic pocket that is allosterically coupled to VP35’s key RNA-binding interface. Experimental tests corroborate the predicted pocket and confirm that stabilizing the open form allosterically disrupts RNA binding. These results demonstrate simulations’ power to characterize hidden conformations and dynamics, uncovering cryptic pockets and allostery that present new therapeutic opportunities.

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