scholarly journals Ribonucleocapsid Formation of Severe Acute Respiratory Syndrome Coronavirus through Molecular Action of the N-Terminal Domain of N Protein

2007 ◽  
Vol 81 (8) ◽  
pp. 3913-3921 ◽  
Author(s):  
Kumar Singh Saikatendu ◽  
Jeremiah S. Joseph ◽  
Vanitha Subramanian ◽  
Benjamin W. Neuman ◽  
Michael J. Buchmeier ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Solution Structure ◽  
Rna Binding ◽  
Crystal Packing ◽  
Rna Recognition ◽  
Viral Membrane ◽  
Ribonucleoprotein Complex ◽  
N Protein ◽  
Cubic Form ◽  
Terminal Domain

ABSTRACT Conserved among all coronaviruses are four structural proteins: the matrix (M), small envelope (E), and spike (S) proteins that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in the lumen. The N-terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding, while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C terminus of the N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17 Å (monoclinic) and at 1.85 Å (cubic), respectively, resolved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the β-sheet core. The functionally important positively charged β-hairpin protrudes out of the core, is oriented similarly to that in the IBV N-NTD, and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggests a common mode of RNA recognition, but they probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs suggests that they use different modes of both RNA recognition and oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

scholarly journals In-silico homology assisted identification of inhibitor of RNA binding against 2019-nCoV N-protein (N terminal domain)

2020 ◽  
pp. 1-9 ◽  
Author(s):  
Phulen Sarma ◽  
Nishant Shekhar ◽  
Manisha Prajapat ◽  
Pramod Avti ◽  
Hardeep Kaur ◽  
...  
Keyword(s):  
In Silico ◽  
Rna Binding ◽  
N Protein ◽  
Terminal Domain

scholarly journals Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein

2020 ◽  
Vol 16 (12) ◽  
pp. e1009100 ◽  
Author(s):  
Dhurvas Chandrasekaran Dinesh ◽  
Dominika Chalupska ◽  
Jan Silhan ◽  
Eliska Koutna ◽  
Radim Nencka ◽  
...  
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Rna Virus ◽  
Structural Basis ◽  
Rna Recognition ◽  
Nonstructural Proteins ◽  
Viral Membrane ◽  
Intrinsically Disordered ◽  
Binding Cleft ◽  
Rna Complex

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.

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

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.

Solution structure of the RNA-binding cold-shock domain of the Chlamydomonas reinhardtii NAB1 protein and insights into RNA recognition

2015 ◽  
Vol 469 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Anne L. Sawyer ◽  
Michael J. Landsberg ◽  
Ian L. Ross ◽  
Olaf Kruse ◽  
Mehdi Mobli ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Solution Structure ◽  
Cold Shock ◽  
Rna Binding ◽  
Specific Binding ◽  
Consensus Sequence ◽  
Rna Recognition ◽  
Nucleic Acid Binding ◽  
Cold Shock Domain ◽  
Higher Eukaryotes

We present the solution structure of the Chlamydomonas reinhardtii nucleic acid-binding protein 1 (NAB1) cold-shock domain (CSD) and compare this to homologues from bacteria and higher eukaryotes. Sequence-specific binding of the CSD consensus sequence (CSDCS) RNA element is also investigated.

scholarly journals Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription

2006 ◽  
Vol 400 (3) ◽  
pp. 449-456 ◽  
Author(s):  
Jampani N. Rao ◽  
Liane Neumann ◽  
Sabine Wenzel ◽  
Kristian Schweimer ◽  
Paul Rösch ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Solution Structure ◽  
Rna Binding ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Recognition Motif ◽  
Transcription Elongation Factor ◽  
Tar Rna

The elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and negative factors. The cellular negative transcription elongation factor NELF (negative elongation factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has α-helical and β-strand elements adopting a βαββαβ fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with Kd values in the low-micromolar range.

scholarly journals Multiple Nucleic Acid Binding Sites and Intrinsic Disorder of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Implications for Ribonucleocapsid Protein Packaging

2008 ◽  
Vol 83 (5) ◽  
pp. 2255-2264 ◽  
Author(s):  
Chung-Ke Chang ◽  
Yen-Lan Hsu ◽  
Yuan-Hsiang Chang ◽  
Fa-An Chao ◽  
Ming-Chya Wu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Intrinsic Disorder ◽  
Scattering Data ◽  
Nucleic Acid Binding ◽  
Structural Domains ◽  
N Protein ◽  
Disordered Regions

ABSTRACT The nucleocapsid protein (N) of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genomic RNA and is crucial for viability. However, the RNA-binding mechanism is poorly understood. We have shown previously that the N protein contains two structural domains—the N-terminal domain (NTD; residues 45 to 181) and the C-terminal dimerization domain (CTD; residues 248 to 365)—flanked by long stretches of disordered regions accounting for almost half of the entire sequence. Small-angle X-ray scattering data show that the protein is in an extended conformation and that the two structural domains of the SARS-CoV N protein are far apart. Both the NTD and the CTD have been shown to bind RNA. Here we show that all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively. The effect can be explained by the “coupled-allostery” model, devised to explain the allosteric effect in a multidomain regulatory system. Although the N proteins of different coronaviruses share very low sequence homology, the physicochemical features described above may be conserved across different groups of Coronaviridae. The current results underscore the important roles of multisite nucleic acid binding and intrinsic disorder in N protein function and RNP packaging.

scholarly journals The solution structure of Dead End bound to AU-rich RNA reveals an unprecedented mode of tandem RRM-RNA recognition required for mRNA regulation

2019 ◽  
Author(s):  
Malgorzata M. Duszczyk ◽  
Harry Wischnewski ◽  
Tamara Kazeeva ◽  
Fionna E. Loughlin ◽  
Christine von Schroetter ◽  
...  
Keyword(s):  
Solution Structure ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Target Selection ◽  
Binding Pocket ◽  
Rna Recognition ◽  
Sequence Motifs ◽  
Germline Development ◽  
Dead End ◽  
Recognition Motifs

AbstractDead End (DND1) is an RNA-binding protein essential for germline development through its role in post-transcriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1’s tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA. The structure reveals how an NYAYUNN element is specifically recognized, reconciling seemingly contradictory sequence motifs discovered in recent genome-wide studies. RRM1 acts as a main binding platform, including atypical extensions to the canonical RRM fold. RRM2 acts cooperatively with RRM1, capping the RNA using an unusual binding pocket, leading to an unprecedented mode of tandem RRM-RNA recognition. We show that the consensus motif is sufficient to mediate upregulation of a reporter gene in human cells and that this process depends not only on RNA binding by the RRMs, but also on DND1’s double-stranded RNA binding domain (dsRBD), which is dispensable for target binding in cellulo. Our results point to a model where DND1 target selection is mediated by a non-canonical mode of AU-rich RNA recognition by the tandem RRMs and a role for the dsRBD in the recruitment of effector complexes responsible for target regulation.

scholarly journals Expression and analysis of the SAM-dependent RNA methyltransferase Rsm22 from Saccharomyces cerevisiae

2021 ◽  
Vol 77 (6) ◽  
Author(s):  
Jahangir Alam ◽  
Farah Tazkera Rahman ◽  
Shiv K. Sah-Teli ◽  
Rajaram Venkatesan ◽  
M. Kristian Koski ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Solution Structure ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Information ◽  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Ribosomal Subunit ◽  
Structural Motif ◽  
Terminal Domain

The Saccharomyces cerevisiae Rsm22 protein (Sc-Rsm22), encoded by the nuclear RSM22 (systematic name YKL155c) gene, is a distant homologue of Rsm22 from Trypanosoma brucei (Tb-Rsm22) and METTL17 from mouse (Mm-METTL17). All three proteins have been shown to be associated with mitochondrial gene expression, and Sc-Rsm22 has been documented to be essential for mitochondrial respiration. The Sc-Rsm22 protein comprises a polypeptide of molecular weight 72.2 kDa that is predicted to harbor an N-terminal mitochondrial targeting sequence. The precise physiological function of Rsm22-family proteins is unknown, and no structural information has been available for Sc-Rsm22 to date. In this study, Sc-Rsm22 was expressed and purified in monomeric and dimeric forms, their folding was confirmed by circular-dichroism analyses and their low-resolution structures were determined using a small-angle X-ray scattering (SAXS) approach. The solution structure of the monomeric form of Sc-Rsm22 revealed an elongated three-domain arrangement, which differs from the shape of Tb-Rsm22 in its complex with the mitochondrial small ribosomal subunit in T. brucei (PDB entry 6sg9). A bioinformatic analysis revealed that the core domain in the middle (Leu117–Asp462 in Sc-Rsm22) resembles the corresponding region in Tb-Rsm22, including a Rossmann-like methyltransferase fold followed by a zinc-finger-like structure. The latter structure is not present in this position in other methyltransferases and is therefore a unique structural motif for this family. The first half of the C-terminal domain is likely to form an OB-fold, which is typically found in RNA-binding proteins and is also seen in the Tb-Rsm22 structure. In contrast, the N-terminal domain of Sc-Rsm22 is predicted to be fully α-helical and shares no sequence similarity with other family members. Functional studies demonstrated that the monomeric variant of Sc-Rsm22 methylates mitochondrial tRNAs in vitro. These data suggest that Sc-Rsm22 is a new and unique member of the RNA methyltransferases that is important for mitochondrial protein synthesis.

scholarly journals Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein

2005 ◽  
Vol 86 (12) ◽  
pp. 3303-3310 ◽  
Author(s):  
Jaehwan You ◽  
Brian K. Dove ◽  
Luis Enjuanes ◽  
Marta L. DeDiego ◽  
Enrique Alvarez ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Subcellular Localization ◽  
Rna Binding ◽  
Cell Signalling ◽  
Bioinformatic Analysis ◽  
Binding Motif ◽  
Nuclear Localization Signals ◽  
N Protein ◽  
Localization Signal ◽  
Infected Cells

The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.

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