scholarly journals Structure of the Paramyxovirus Parainfluenza Virus 5 Nucleoprotein in Complex with an Amino-Terminal Peptide of the Phosphoprotein

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Megha Aggarwal ◽  
George P. Leser ◽  
Christopher A. Kors ◽  
Robert A. Lamb
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Rna Binding ◽  
Hinge Region ◽  
Parainfluenza Virus ◽  
Free Form ◽  
Conformational Switching ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Parainfluenza Virus 5

ABSTRACT Parainfluenza virus 5 (PIV5) belongs to the family Paramyxoviridae , which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N 0 P) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 N 0 P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N 0 P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication. IMPORTANCE Paramyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.

scholarly journals The crystal structure of the Rv0301-Rv0300 VapBC-3 toxin-antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA-binding site

2012 ◽  
Vol 21 (11) ◽  
pp. 1754-1767 ◽  
Author(s):  
Andrew B. Min ◽  
Linda Miallau ◽  
Michael R. Sawaya ◽  
Jeff Habel ◽  
Duilio Cascio ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Active Site ◽  
Rna Binding

scholarly journals Recombinant SARS-CoV-2 Nucleocapsid Protein: Expression, Purification, and Its Biochemical Characterization and Utility in Serological Assay Development to Assess Immunological Responses to SARS-CoV-2 Infection

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1039
Author(s):  
Da Di ◽  
Mythili Dileepan ◽  
Shamim Ahmed ◽  
Yuying Liang ◽  
Hinh Ly
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Biochemical Characterization ◽  
Free Form ◽  
Bacterial Cells ◽  
Serum Samples ◽  
Mobility Shift ◽  
Independent Manner ◽  
Serological Assay ◽  
Terminal Domain

The SARS-CoV-2 nucleocapsid protein (N) binds a single-stranded viral RNA genome to form a helical ribonucleoprotein complex that is packaged into virion particles. N is relatively conserved among coronaviruses and consists of the N-terminal domain (NTD) and C-terminal domain (CTD), which are flanked by three disorganized regions. N is highly immunogenic and has been widely used to develop a serological assay as a diagnostic tool for COVID-19 infection, although there is a concern that the natural propensity of N to associate with RNA might compromise the assay’s specificity. We expressed and purified from bacterial cells two recombinant forms of SARS-CoV-2 N, one from the soluble fraction of bacterial cell lysates that is strongly associated with bacterial RNAs and the other that is completely devoid of RNAs. We showed that both forms of N can be used to develop enzyme-linked immunosorbent assays (ELISAs) for the specific detection of human and mouse anti-N monoclonal antibodies (mAb) as well as feline SARS-CoV-2 seropositive serum samples, but that the RNA-free form of N exhibits a slightly higher level of sensitivity than the RNA-bound form to react to anti-N mouse mAb. Using the electrophoretic mobility shift assay (EMSA), we also showed that N preferentially binds ssRNA in a sequence-independent manner and that both NTD and CTD of N contribute to RNA-binding activity. Collectively, our study describes methods to express, purify, and biochemically characterize the SARS-CoV-2 N protein and to use it for the development of serological assays to detect SARS-CoV-2 infection.

scholarly journals Crystal Structure of the YchF Protein Reveals Binding Sites for GTP and Nucleic Acid

2003 ◽  
Vol 185 (14) ◽  
pp. 4031-4037 ◽  
Author(s):  
Alexey Teplyakov ◽  
Galina Obmolova ◽  
Seung Y. Chu ◽  
John Toedt ◽  
Edward Eisenstein ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Binding Site ◽  
Coiled Coil ◽  
Genomic Analysis ◽  
Protein Fluorescence ◽  
Terminal Domain ◽  
Fluorescence Measurements ◽  
Α Helix

ABSTRACT The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 Å resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced α-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an α-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.

scholarly journals On the Stability of Parainfluenza Virus 5 F Proteins

2015 ◽  
Vol 89 (6) ◽  
pp. 3438-3441 ◽  
Author(s):  
Taylor A. Poor ◽  
Albert S. Song ◽  
Brett D. Welch ◽  
Christopher A. Kors ◽  
Theodore S. Jardetzky ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Conformational Change ◽  
Point Mutations ◽  
Parainfluenza Virus ◽  
Hydrophobic Pocket ◽  
F Protein ◽  
Conformational Effects ◽  
Parainfluenza Virus 5 ◽  
The Stability

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.

scholarly journals Structure of anEscherichia coliHfq:RNA complex at 0.97 Å resolution

2014 ◽  
Vol 70 (11) ◽  
pp. 1492-1497 ◽  
Author(s):  
Eike C. Schulz ◽  
Orsolya Barabas
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Binding Site ◽  
Small Rnas ◽  
Target Genes ◽  
Rna Binding ◽  
Base Pairing ◽  
Rna Chaperone ◽  
Target Mrnas ◽  
Rna Interaction

In bacteria, small RNAs (sRNAs) silence or activate target genes through base pairing with the mRNA, thereby modulating its translation. A central player in this process is the RNA chaperone Hfq, which facilitates the annealing of sRNAs with their target mRNAs. Hfq has two RNA-binding surfaces that recognize A-rich and U-rich sequences, and is believed to bind an sRNA–mRNA pair simultaneously. However, how Hfq promotes annealing remains unclear. Here, the crystal structure ofEscherichia coliHfq is presented in complex with U6-RNA bound to its proximal binding site at 0.97 Å resolution, revealing the Hfq–RNA interaction in exceptional detail.

scholarly journals Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

2020 ◽  
Author(s):  
Sisi Kang ◽  
Mei Yang ◽  
Zhongsi Hong ◽  
Liping Zhang ◽  
Zhaoxia Huang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Structural Information ◽  
Antiviral Drug ◽  
Binding Domain ◽  
Binding Studies ◽  
Terminal Domain ◽  
Rna Binding Domain

AbstractThe outbreak of coronavirus disease (COVID-19) in China caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. It is currently no specific viral protein targeted therapeutics yet. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein is yet to be clear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although overall structure is similar with other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the β-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

scholarly journals Crystal Structure of Human Cyclophilin A Bound to the Amino-Terminal Domain of HIV-1 Capsid

Cell ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 1285-1294 ◽  
Author(s):  
Theresa R Gamble ◽  
Felix F Vajdos ◽  
Sanghee Yoo ◽  
David K Worthylake ◽  
Megan Houseweart ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cyclophilin A ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain ◽  
Hiv 1

Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase

Nature ◽  
10.1038/18050 ◽  
1999 ◽  
Vol 398 (6722) ◽  
pp. 84-90 ◽  
Author(s):  
Wuyi Meng ◽  
Sansana Sawasdikosol ◽  
Steven J. Burakoff ◽  
Michael J. Eck
Keyword(s):  
Binding Site ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

scholarly journals Novel Structure and Unexpected RNA-Binding Ability of the C-Terminal Domain of Herpes Simplex Virus 1 Tegument Protein UL21

2016 ◽  
Vol 90 (12) ◽  
pp. 5759-5769 ◽  
Author(s):  
Claire M. Metrick ◽  
Ekaterina E. Heldwein
Keyword(s):  
Crystal Structure ◽  
Herpes Simplex ◽  
Rna Binding ◽  
Tegument Protein ◽  
Functional Importance ◽  
Binding Ability ◽  
Herpes Simplex Virus 1 ◽  
3 Dimensional ◽  
Terminal Domain ◽  
Simplex Virus

ABSTRACTProteins forming the tegument layers of herpesviral virions mediate many essential processes in the viral replication cycle, yet few have been characterized in detail. UL21 is one such multifunctional tegument protein and is conserved among alphaherpesviruses. While UL21 has been implicated in many processes in viral replication, ranging from nuclear egress to virion morphogenesis to cell-cell spread, its precise roles remain unclear. Here we report the 2.7-Å crystal structure of the C-terminal domain of herpes simplex virus 1 (HSV-1) UL21 (UL21C), which has a unique α-helical fold resembling a dragonfly. Analysis of evolutionary conservation patterns and surface electrostatics pinpointed four regions of potential functional importance on the surface of UL21C to be pursued by mutagenesis. In combination with the previously determined structure of the N-terminal domain of UL21, the structure of UL21C provides a 3-dimensional framework for targeted exploration of the multiple roles of UL21 in the replication and pathogenesis of alphaherpesviruses. Additionally, we describe an unanticipated ability of UL21 to bind RNA, which may hint at a yet unexplored function.IMPORTANCEDue to the limited genomic coding capacity of viruses, viral proteins are often multifunctional, which makes them attractive antiviral targets. Such multifunctionality, however, complicates their study, which often involves constructing and characterizing null mutant viruses. Systematic exploration of these multifunctional proteins requires detailed road maps in the form of 3-dimensional structures. In this work, we determined the crystal structure of the C-terminal domain of UL21, a multifunctional tegument protein that is conserved among alphaherpesviruses. Structural analysis pinpointed surface areas of potential functional importance that provide a starting point for mutagenesis. In addition, the unexpected RNA-binding ability of UL21 may expand its functional repertoire. The structure of UL21C and the observation of its RNA-binding ability are the latest additions to the navigational chart that can guide the exploration of the multiple functions of UL21.

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