Probing the Impact of the echinT C-Terminal Domain on Structure and Catalysis

2010 ◽  
Vol 404 (4) ◽  
pp. 627-638 ◽  
Author(s):  
Sanaa Bardaweel ◽  
James Pace ◽  
Tsui-Fen Chou ◽  
Vivian Cody ◽  
Carston R. Wagner
Keyword(s):  
Terminal Domain ◽  
The Impact

scholarly journals The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum

2016 ◽  
Vol 1858 (1) ◽  
pp. 130-138 ◽  
Author(s):  
Yoshitaka Nakayama ◽  
Michael Becker ◽  
Haleh Ebrahimian ◽  
Tomoyuki Konishi ◽  
Hisashi Kawasaki ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Terminal Domain ◽  
The Impact

scholarly journals Nuclear Import of Adenovirus DNA Involves Direct Interaction of Hexon with an N-Terminal Domain of the Nucleoporin Nup214

2014 ◽  
Vol 89 (3) ◽  
pp. 1719-1730 ◽  
Author(s):  
Aurélia Cassany ◽  
Jessica Ragues ◽  
Tinglu Guan ◽  
Dominique Bégu ◽  
Harald Wodrich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Nuclear Import ◽  
Viral Genome ◽  
Molecular Basis ◽  
Nuclear Pore ◽  
Viral Dna ◽  
Terminal Domain ◽  
Cytoplasmic Face ◽  
The Impact

ABSTRACTIn this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Bothin vitrohexon binding andin vivonuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV.IMPORTANCEAdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.

scholarly journals The Impact of the Human DNA Topoisomerase II C-Terminal Domain on Activity

PLoS ONE ◽  
2008 ◽  
Vol 3 (3) ◽  
pp. e1754 ◽  
Author(s):  
Emma L. Meczes ◽  
Kathryn L. Gilroy ◽  
Katherine L. West ◽  
Caroline A. Austin
Keyword(s):  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Terminal Domain ◽  
Human Dna ◽  
The Impact

scholarly journals Structurally Guided Removal of DeISGylase Biochemical Activity from Papain-Like Protease Originating from Middle East Respiratory Syndrome Coronavirus

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Courtney M. Daczkowski ◽  
Octavia Y. Goodwin ◽  
John V. Dzimianski ◽  
Jonathan J. Farhat ◽  
Scott D. Pegan
Keyword(s):  
Middle East ◽  
Gene Product ◽  
Isothermal Calorimetry ◽  
Middle East Respiratory Syndrome ◽  
Human Interferon ◽  
Type I ◽  
Peptide Cleavage ◽  
Terminal Domain ◽  
Immune Pathways ◽  
The Impact

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen that is the causative agent for Middle East respiratory syndrome (MERS). With MERS outbreaks resulting in over 35% fatalities and now spread to 27 countries, MERS-CoV poses a significant ongoing threat to global human health. As part of its viral genome, MERS-CoV encodes a papain-like protease (PLpro) that has been observed to act as a deubiquitinase and deISGylase to antagonize type I interferon (IFN-I) immune pathways. This activity is in addition to its viral polypeptide cleavage function. Although the overall impact of MERS-CoV PLpro function is observed to be essential, difficulty has been encountered in delineating the importance of its separate functions, particularly its deISGylase activity. As a result, the interface of MERS-CoV and human interferon-stimulated gene product 15 (hISG15) was probed with isothermal calorimetry, which suggests that the C-terminal domain of hISG15 is principally responsible for interactions. Subsequently, the structure of MERS-CoV PLpro was solved to 2.4 Å in complex with the C-terminal domain of hISG15. Utilizing this structural information, mutants were generated that lacked appreciable deISGylase activity but retained wild-type deubiquitinase and peptide cleavage activities. Hence, this provides a new platform for understanding viral deISGylase activity within MERS-CoV and other CoVs. IMPORTANCE Coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), encode a papain-like protease (PLpro) that possesses the ability to antagonize interferon immune pathways through the removal of ubiquitin and interferon-stimulated gene product 15 (ISG15) from target proteins. The lack of CoV proteases with attenuated deISGylase activity has been a key obstacle in delineating the impact between deubiquitinase and deISGylase activities on viral host evasion and pathogenesis. Here, biophysical techniques revealed that MERS-CoV PLpro chiefly engages human ISG15 through its C-terminal domain. The first structure of MERS-CoV PLpro in complex with this domain exposed the interface between these two entities. Employing these structural insights, mutations were employed to selectively remove deISGylase activity with no appreciable impact on its other deubiquitinase and peptide cleavage biochemical properties. Excitingly, this study introduces a new tool to probe the pathogenesis of MERS-CoV and related viruses through the removal of viral deISGylase activity.

scholarly journals Clinical and Functional Consequences of C-Terminal Variants in MCT8: A Case Series

2020 ◽  
Author(s):  
Ferdy S van Geest ◽  
Marcel E Meima ◽  
Kyra E Stuurman ◽  
Nicole I Wolf ◽  
Marjo S van der Knaap ◽  
...  
Keyword(s):  
Clinical Features ◽  
Genetic Variants ◽  
Transmembrane Domain ◽  
Residual Activity ◽  
Case Series ◽  
Thyroid Function Tests ◽  
Terminal Domain ◽  
Functional Consequences ◽  
Mct8 Deficiency ◽  
The Impact

Abstract Context Genetic variants in SLC16A2, encoding the thyroid hormone transporter MCT8, can cause intellectual and motor disability and abnormal serum thyroid function tests, known as MCT8 deficiency. The C-terminal domain of MCT8 is poorly conserved, which complicates prediction of the deleteriousness of variants in this region. We studied the functional consequences of 5 novel variants within this domain and their relation to the clinical phenotypes. Methods We enrolled male subjects with intellectual disability in whom genetic variants were identified in exon 6 of SLC16A2. The impact of identified variants was evaluated in transiently transfected cell lines and patient-derived fibroblasts. Results Seven individuals from 5 families harbored potentially deleterious variants affecting the C-terminal domain of MCT8. Two boys with clinical features considered atypical for MCT8 deficiency had a missense variant [c.1724A>G;p.(His575Arg) or c.1796A>G;p.(Asn599Ser)] that did not affect MCT8 function in transfected cells or patient-derived fibroblasts, challenging a causal relationship. Two brothers with classical MCT8 deficiency had a truncating c.1695delT;p.(Val566*) variant that completely inactivated MCT8 in vitro. The 3 other boys had relatively less-severe clinical features and harbored frameshift variants that elongate the MCT8 protein [c.1805delT;p.(Leu602HisfsTer680) and c.del1826-1835;p.(Pro609GlnfsTer676)] and retained ~50% residual activity. Additional truncating variants within transmembrane domain 12 were fully inactivating, whereas those within the intracellular C-terminal tail were tolerated. Conclusions Variants affecting the intracellular C-terminal tail of MCT8 are likely benign unless they cause frameshifts that elongate the MCT8 protein. These findings provide clinical guidance in the assessment of the pathogenicity of variants within the C-terminal domain of MCT8.

scholarly journals The Impact of the C-Terminal Region on the Interaction of Topoisomerase II Alpha with Mitotic Chromatin

2019 ◽  
Vol 20 (5) ◽  
pp. 1238 ◽  
Author(s):  
Melissa Antoniou-Kourounioti ◽  
Michael Mimmack ◽  
Andrew Porter ◽  
Christine Farr
Keyword(s):  
Topoisomerase Ii ◽  
Subcellular Localisation ◽  
Post Translational Modification ◽  
Topoisomerase Ii Alpha ◽  
Terminal Domain ◽  
Anaphase Onset ◽  
Specific Behaviour ◽  
Mitotic Phosphorylation ◽  
The Impact ◽  
Mitotic Chromatin

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme’s residence time on mitotic chromatin, as well as its subcellular localisation.

scholarly journals Selective Disruption of SERINC5 Antagonism by Nef Impairs SIV Replication in Primary CD4+ T Cells

2021 ◽  
Author(s):  
Sanath Kumar Janaka ◽  
Alexandra V Palumbo ◽  
Aidin Tavakoli-Tameh ◽  
David T Evans
Keyword(s):  
T Cells ◽  
Rhesus Macaque ◽  
Transmembrane Protein ◽  
Viral Infectivity ◽  
Class I Mhc ◽  
Systematic Analysis ◽  
Mhc I ◽  
Terminal Domain ◽  
The Impact ◽  
Hiv 1

The Nef proteins of HIV-1 and SIV enhance viral infectivity by preventing the incorporation of the multipass transmembrane protein serine incorporator 5 (SERINC5), and to a lesser extent SERINC3, into virions. In addition to counteracting SERINCs, SIV Nef also downmodulates several transmembrane proteins from the surface of virus-infected cells, including simian tetherin, CD4 and MHC class I (MHC I) molecules. From a systematic analysis of alanine substitutions throughout the SIVmac239 Nef protein, we identified residues that are required to counteract SERINC5. This information was used to engineer an infectious molecular clone of SIV (SIVmac239nefAV), which differs by two amino acids in the N-terminal domain of Nef that make the virus sensitive to SERINC5 while retaining other activities of Nef. SIVmac239nefAV downmodulates CD3, CD4, MHC I and simian tetherin, but cannot counteract SERINC5. In primary rhesus macaque CD4+ T cells, SIVmac239nefAV exhibits impaired infectivity and replication compared to wild-type SIVmac239. These results demonstrate that SERINC5 antagonism can be separated from other Nef functions and reveal the impact of SERINC5 on lentiviral replication. Importance: SERINC5, a multipass transmembrane protein, is incorporated into retroviral particles during assembly. This leads to a reduction of particle infectivity by inhibiting virus fusion with the target cell membrane. The Nef proteins of HIV-1 and SIV enhance viral infectivity by preventing the incorporation of SERINC5 into virions. However, the relevance of this restriction factor in viral replication has not been elucidated. Here we report a systematic mapping of Nef residues required for SERINC5 antagonism. Counter screens for three other functions of Nef helped identify two residues in the N-terminal domain of Nef, which when mutated make Nef selectively susceptible to SERINC5. Since Nef is multi-functional, genetic separation of SERINC5 antagonism from its other functions affords comparison of the replication of isogenic viruses that are or are not sensitive to SERINC5. Such a strategy revealed the impact of SERINC5 on SIV replication in primary rhesus macaque CD4+ T-cells.

scholarly journals Molecular basis of immune evasion by the delta and kappa SARS-CoV-2 variants

2021 ◽  
Author(s):  
Matthew McCallum ◽  
Alexandra C Walls ◽  
Kaitlin R Sprouse ◽  
John E Bowen ◽  
Laura Rosen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Evasion ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Terminal Domain ◽  
Structural Framework ◽  
Ancestral Virus ◽  
Delta Receptor ◽  
The Impact

Worldwide SARS-CoV-2 transmission leads to the recurrent emergence of variants, such as the recently described B.1.617.1 (kappa), B.1.617.2 (delta) and B.1.617.2+ (delta+). The B.1.617.2 (delta) variant of concern is causing a new wave of infections in many countries, mostly affecting unvaccinated individuals, and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing the impact of individual mutations on immune evasion. Mutations in the B.1.617.1 (kappa) and B.1.617.2 (delta) spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including an unexpected remodeling of the B.1.617.2 (delta) N-terminal domain. The binding affinity of the B.1.617.1 (kappa) and B.1.617.2 (delta) receptor-binding domain for ACE2 is comparable to the ancestral virus whereas B.1.617.2+ (delta+) exhibits markedly reduced affinity. We describe a previously uncharacterized class of N-terminal domain-directed human neutralizing monoclonal antibodies cross-reacting with several variants of concern, revealing a possible target for vaccine development.

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