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.

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 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 Deamidation in Moxetumomab Pasudotox Leading to Conformational Change and Immunotoxin Activity Loss

2019 ◽  
Author(s):  
X. Lu ◽  
S. Lin ◽  
N. De Mel ◽  
A. Parupudi ◽  
M. Pandey ◽  
...  
Keyword(s):  
Biological Activity ◽  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Scanning Calorimetry ◽  
Exchange Mass Spectrometry ◽  
Binding Interface ◽  
Hydrogen Deuterium ◽  
Asparagine Deamidation ◽  
Deuterium Exchange Mass Spectrometry

ABSTRACTAsparagine deamidation is a common posttranslational modification in which asparagine is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kilodalton (kDa) truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which suggests a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide a possible explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest an approach that can be used for process control to ensure product quality and process consistency.Statement of SignificanceAsparagine deamidation can have a potentially significant impact on protein therapeutics. Previous studies have revealed that deamidation at a single site significantly reduces the biological activity of moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin developed for the treatment of B-cell malignancies. Surprisingly, despite the fact that deamidation introduced a negative charge, the deamidated variant eluted earlier than its unmodified form on anion exchange chromatography. In order to understand these observations, we isolated the deamidated variant using an anion exchange column and characterized it by differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry. The results revealed the conformational change caused by the deamidation, which explains the diminished biological activity of the variant and its early elution time on anion exchange chromatography.

The unresolved puzzle why alanine extensions cause disease

2013 ◽  
Vol 394 (8) ◽  
pp. 951-963 ◽  
Author(s):  
Reno Winter ◽  
Jens Liebold ◽  
Elisabeth Schwarz
Keyword(s):  
Old Age ◽  
Protein Misfolding ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Binding Protein ◽  
Biochemical Properties ◽  
Fibril Formation ◽  
Terminal Domain ◽  
Skeletal Malformations ◽  
Biochemical Analyses

Abstract The prospective increase in life expectancy will be accompanied by a rise in the number of elderly people who suffer from ill health caused by old age. Many diseases caused by aging are protein misfolding diseases. The molecular mechanisms underlying these disorders receive constant scientific interest. In addition to old age, mutations also cause congenital protein misfolding disorders. Chorea Huntington, one of the most well-known examples, is caused by triplet extensions that can lead to more than 100 glutamines in the N-terminal region of huntingtin, accompanied by huntingtin aggregation. So far, nine disease-associated triplet extensions have also been described for alanine codons. The extensions lead primarily to skeletal malformations. Eight of these proteins represent transcription factors, while the nuclear poly-adenylate binding protein 1, PABPN1, is an RNA binding protein. Additional alanines in PABPN1 lead to the disease oculopharyngeal muscular dystrophy (OPMD). The alanine extension affects the N-terminal domain of the protein, which has been shown to lack tertiary contacts. Biochemical analyses of the N-terminal domain revealed an alanine-dependent fibril formation. However, fibril formation of full-length protein did not recapitulate the findings of the N-terminal domain. Fibril formation of intact PABPN1 was independent of the alanine segment, and the fibrils displayed biochemical properties that were completely different from those of the N-terminal domain. Although intranuclear inclusions have been shown to represent the histochemical hallmark of OPMD, their role in pathogenesis is currently unclear. Several cell culture and animal models have been generated to study the molecular processes involved in OPMD. These studies revealed a number of promising future therapeutic strategies that could one day improve the quality of life for the patients.

scholarly journals ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer

2019 ◽  
Author(s):  
Xiaoshan Shi ◽  
Adam L. Yokom ◽  
Chunxin Wang ◽  
Lindsey N. Young ◽  
Richard J. Youle ◽  
...  
Keyword(s):  
Single Molecule ◽  
Exchange Mass Spectrometry ◽  
Terminal Domain ◽  
Negative Stain ◽  
Complex Organization ◽  
Hydrogen Deuterium ◽  
Negative Stain Electron Microscopy ◽  
Deuterium Exchange Mass Spectrometry ◽  
Structure Similarity ◽  
20 Nm

AbstractThe autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain electron microscopy (EM) and multiangle light scattering showed that FIP200 is a dimer whilst a single molecule each of the other subunits is present. The FIP200 NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ~20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13-ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structure similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy initiating TBK1 kinase and the ULK1 kinase complex.SummaryThe human ULK complex consists of ULK1/2, FIP200, ATG13, and ATG101. We found that the FIP200 N-terminal domain is a C-shaped dimer that binds directly to a single ATG13 molecule and serves as the organizing hub of the complex.

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