Interdomain salt‐bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization

Jeevan, B.; Johnson, Kristen A.; Husby, Monica L.; Frick, Cary T.; Gerstman, Bernard S.; Stahelin, Robert V.; Chapagain, Prem P.

doi:10.1002/pro.2969

Cited by 23 publications

(30 citation statements)

References 40 publications

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“…(C)]. Lastly we investigated plausible salt bridges formed at the interface of ARID and PHD1 domains, because salt bridges are frequently observed at transient domain‐domain interfaces . Since we had previously performed 5 parallel runs, we only selected the salt‐bridges observed in no less than two runs.…”

Section: Resultsmentioning

confidence: 99%

Cofactors-loaded quaternary structure of lysine-specific demethylase 5C (KDM5C) protein: Computational model

Peng

Alexov

2016

Proteins

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The KDM5C gene (also known as JARID1C and SMCX) is located on the X chromosome and encodes a ubiquitously expressed 1,560-aa protein, which plays an important role in lysine methylation (specifically reverses tri- and di-methylation of Lys4 of histone H3). Currently, thirteen missense mutations in KDM5C have been linked to X-linked mental retardation. However, the molecular mechanism of disease is currently unknown due to the experimental difficulties in expressing such large protein and the lack of experimental 3D structure. In this work, we utilize homology modeling, docking, and experimental data to predict 3D structures of KDM5C domains and their mutual arrangement. The resulting quaternary structure includes KDM5C JmjN, ARID, PHD1, JmjC, ZF domains, substrate histone peptide, enzymatic cofactors and DNA. The predicted quaternary structure was investigated with molecular dynamic simulation for its stability, and further analysis was carried out to identify features measured experimentally. The predicted structure of KDM5C was used to investigate the effects of disease-causing mutations and it was shown that the mutations alter domain stability and inter-domain interactions. The structural model reported in this work could prompt experimental investigations of KDM5C domain-domain interaction and exploration of undiscovered functionalities.

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Section: Resultsmentioning

confidence: 99%

Cofactors-loaded quaternary structure of lysine-specific demethylase 5C (KDM5C) protein: Computational model

Peng

Alexov

2016

Proteins

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“…Finally, we showed that WWP1-WT ubiquitinated eVP40 and that the presence of higher-molecular-weight (MW) oligomers of eVP40 was reduced significantly in the presence of WWP1-WT but not in the presence of WWP1-C890A. These findings indicate that a functional interaction between eVP40 and WWP1 leads to eVP40 ubiquitination, likely promoting more efficient self-assembly, maturation, and scission of eVP40 VLPs from the plasma membrane (58)(59)(60)(61).…”

mentioning

confidence: 82%

Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

Han

Sagum

Takizawa

et al. 2017

J Virol

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Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of highermolecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress.IMPORTANCE Ebola virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here, we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPXY-host WW domain-mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress.KEYWORDS E3 ubiquitin ligase, L-domain, PPXY, VLPs, VP40, WW domain, WWP1, budding, Ebola virus

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“…The identity of the mutants was concealed during imaging to minimize bias. Heterologous expression levels were confirmed via western blot as previously described (59).…”

Section: Cell Culture and Cell Imagingmentioning

confidence: 99%

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine

Vecchio

Frick

Jeevan

et al. 2018

Journal of Biological Chemistry

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134

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Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causeshemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or a RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures (i.e. monomer, dimer, and octamer) can interact with PScontaining membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys-224, 225 and Lys-274, 275). Substitutions of these residues with alanine reduced PSvesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest http://www.jbc.org/cgi

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Interdomain salt‐bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization

Cited by 23 publications

References 40 publications

Cofactors-loaded quaternary structure of lysine-specific demethylase 5C (KDM5C) protein: Computational model

Cofactors-loaded quaternary structure of lysine-specific demethylase 5C (KDM5C) protein: Computational model

Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine

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