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

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.

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Regulative interactions of the osmosensing C-terminal domain in the trimeric glycine betaine transporter BetP from Corynebacterium glutamicum

Biological Chemistry ◽

10.1515/bc.2009.068 ◽

2009 ◽

Vol 390 (8) ◽

Cited By ~ 10

Author(s):

Reinhard Krämer ◽

Christine Ziegler

Keyword(s):

Amino Acid ◽

Corynebacterium Glutamicum ◽

Protein Interactions ◽

Regulation Mechanism ◽

Protein Protein Interactions ◽

Molecular Switching ◽

X Ray ◽

X Ray Crystallography ◽

Terminal Domain ◽

Domain Reorientation

Abstract Activation of the osmoregulated trimeric betaine transporter BetP from Corynebacterium glutamicum was shown to depend mainly on the correct folding and integrity of its 55 amino acid long, partly α-helical C-terminal domain. Reorientation of the three C-terminal domains in the BetP trimer indicates different lipid-protein and protein-protein interactions of the C-terminal domain during osmoregulation. A regulation mechanism is suggested where this domain switches the transporter from the inactive to the active state. Interpretation of recently obtained electron and X-ray crystallography data of BetP led to a structure-function based model of C-terminal molecular switching involved in osmoregulation.

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3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum : the C-terminal domain is not essential for activity but is required for inhibition by L -serine

Applied Microbiology and Biotechnology ◽

10.1007/s00253-002-1161-y ◽

2002 ◽

Vol 60 (4) ◽

pp. 437-441 ◽

Cited By ~ 39

Author(s):

Peters-Wendisch P. ◽

Netzer R. ◽

Eggeling L. ◽

Sahm H.

Keyword(s):

Corynebacterium Glutamicum ◽

Terminal Domain ◽

Phosphoglycerate Dehydrogenase

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The impact of PHB accumulation on l-glutamate production by recombinant Corynebacterium glutamicum

Journal of Biotechnology ◽

10.1016/j.jbiotec.2007.03.014 ◽

2007 ◽

Vol 132 (3) ◽

pp. 273-279 ◽

Cited By ~ 42

Author(s):

Q LIU ◽

S OUYANG ◽

J KIM ◽

G CHEN

Keyword(s):

Corynebacterium Glutamicum ◽

Glutamate Production ◽

The Impact

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

PLoS ONE ◽

10.1371/journal.pone.0001754 ◽

2008 ◽

Vol 3 (3) ◽

pp. e1754 ◽

Cited By ~ 15

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

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

Journal of Virology ◽

10.1128/jvi.01067-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 16

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.

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

Journal of Molecular Biology ◽

10.1016/j.jmb.2010.09.066 ◽

2010 ◽

Vol 404 (4) ◽

pp. 627-638 ◽

Cited By ~ 14

Author(s):

Sanaa Bardaweel ◽

James Pace ◽

Tsui-Fen Chou ◽

Vivian Cody ◽

Carston R. Wagner

Keyword(s):

Terminal Domain ◽

The Impact

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Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.70.7.3845-3854.2004 ◽

2004 ◽

Vol 70 (7) ◽

pp. 3845-3854 ◽

Cited By ~ 20

Author(s):

Leandro Padilla ◽

Susanne Morbach ◽

Reinhard Krämer ◽

Eduardo Agosin

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Corynebacterium Glutamicum ◽

Glycogen Synthesis ◽

Glycogen Accumulation ◽

E Coli ◽

Wide Range ◽

The Impact ◽

Synthetic Operon ◽

Trehalose Synthesis

ABSTRACT Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.

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

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgaa795 ◽

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.

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