Structural characterization of a C-terminally truncated E5 oncoprotein from papillomavirus in lipid bilayers

2014 ◽  
Vol 395 (12) ◽  
pp. 1443-1452 ◽  
Author(s):  
Dirk Windisch ◽  
Colin Ziegler ◽  
Jochen Bürck ◽  
Anne S. Ulrich
Keyword(s):  
Lipid Bilayers ◽  
Transmembrane Protein ◽  
Growth Factor Receptor ◽  
Helical Conformation ◽  
Resonance Spectroscopy ◽  
Bovine Papillomavirus ◽  
Transmembrane Segments ◽  
C Terminus ◽  
E5 Oncoprotein ◽  
Representative Model

AbstractE5 is the major transforming oncoprotein of bovine papillomavirus, which activates the platelet-derived growth factor receptor β in a highly specific manner. The short transmembrane protein E5 with only 44 residues interacts directly with the transmembrane segments of the receptor, but structural details are not available. Biophysical investigations are challenging, because the hydrophobic E5 protein tends to aggregate and get cross-linked non-specifically via two Cys residues near its C-terminus. Here, we demonstrate that a truncation by 10 amino acids creates a more manageable protein that can be conveniently used for structure analysis. Synchrotron radiation circular dichroism and solid-state15N- and31P-nuclear magnetic resonance spectroscopy show that this E5 variant serves as a representative model for the wild-type protein. The helical conformation of the transmembrane segment, its orientation in the lipid bilayer, and the ability to form homodimers in the membrane are not affected by the C-terminal truncation.

scholarly journals Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF β receptor in an active dimeric conformation

2017 ◽  
Vol 114 (35) ◽  
pp. E7262-E7271 ◽  
Author(s):  
Alexander G. Karabadzhak ◽  
Lisa M. Petti ◽  
Francisco N. Barrera ◽  
Anne P. B. Edwards ◽  
Andrés Moya-Rodríguez ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Receptor Activation ◽  
Bovine Papillomavirus ◽  
Detailed Model ◽  
E5 Protein ◽  
Membrane Modeling ◽  
E5 Oncoprotein ◽  
Β Receptor ◽  
Dynamics Simulations

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex. In this model, an active dimer of the PDGFβR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFβR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFβR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.

scholarly journals Membrane Orientation of the Human Papillomavirus Type 16 E5 Oncoprotein

2009 ◽  
Vol 84 (4) ◽  
pp. 1696-1703 ◽  
Author(s):  
Ewa Krawczyk ◽  
Frank A. Suprynowicz ◽  
Sawali R. Sudarshan ◽  
Richard Schlegel
Keyword(s):  
Human Papillomavirus ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Biologically Active ◽  
Human Papillomavirus Type 16 ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
Low Concentrations ◽  
E5 Protein ◽  
E5 Oncoprotein

ABSTRACT The E5 protein of human papillomavirus type 16 is a small, hydrophobic protein that localizes predominantly to membranes of the endoplasmic reticulum (ER). To define the orientation of E5 in these membranes, we employed a differential, detergent permeabilization technique that makes use of the ability of low concentrations of digitonin to selectively permeabilize the plasma membrane and saponin to permeabilize all cellular membranes. We then generated a biologically active E5 protein that was epitope tagged at both its N and C termini and determined the accessibility of these termini to antibodies in the presence and absence of detergents. In both COS cells and human ectocervical cells, the C terminus of E5 was exposed to the cytoplasm, whereas the N terminus was restricted to the lumen of the ER. Finally, the deletion of the E5 third transmembrane domain (and terminal hydrophilic amino acids) resulted in a protein with its C terminus in the ER lumen. Taken together, these topology findings are compatible with a model of E5 being a 3-pass transmembrane protein and with studies demonstrating its C terminus interacting with cytoplasmic proteins.

scholarly journals Inner-Nuclear-Membrane-Associated Degradation Employs Dfm1-Independent Retrotranslocation and Alleviates Misfolded Transmembrane-Protein Toxicity

2021 ◽  
pp. mbc.E20-11-0720
Author(s):  
Matthew P. Flagg ◽  
Margaret A. Wangeline ◽  
Sarah R. Holland ◽  
Sascha H. Duttke ◽  
Christopher Benner ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Nuclear Membrane ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Extraction Process ◽  
26S Proteasome ◽  
Energetic Cost ◽  
Inner Nuclear Membrane ◽  
Transmembrane Segments ◽  
Protein Toxicity

Prior to their delivery to and degradation by the 26S proteasome, misfolded transmembrane proteins of the ER and inner-nuclear membrane must be extracted from lipid bilayers. This extraction process, known as retrotranslocation, requires both quality-control E3 ubiquitin ligases and dislocation factors that diminish the energetic cost of dislodging the transmembrane segments of a protein. Recently, we showed that retrotranslocation of all ER transmembrane proteins requires the Dfm1 rhomboid pseudoprotease. However, we did not investigate whether Dfm1 also mediated retrotranslocation of transmembrane substrates in the inner-nuclear membrane (INM), which is contiguous with the ER but functionally separated from it by nucleoporins. Here, we show that canonical retrotranslocation occurs during INM-associated degradation (INMAD) but proceeds independently of Dfm1. Despite this independence, ERAD-M and INMAD cooperate to mitigate proteotoxicity. We show a novel misfolded-transmembrane-protein toxicity that elicits genetic suppression, demonstrating the cell's ability to tolerate a toxic burden of misfolded transmembrane proteins without functional INMAD or ERAD-M. This strikingly contrasted the suppression of the dfm1Δ null, which leads to the resumption of ERAD-M through HRD-complex remodeling. Thus, we conclude that INM retrotranslocation proceeds through a novel, private channel, which can be studied by virtue of its role in alleviating membrane-associated proteotoxicity.

scholarly journals The C Terminus of Hepatitis C Virus NS4A Encodes an Electrostatic Switch That Regulates NS5A Hyperphosphorylation and Viral Replication

2007 ◽  
Vol 81 (17) ◽  
pp. 8905-8918 ◽  
Author(s):  
Brett D. Lindenbach ◽  
Béla M. Prágai ◽  
Roland Montserret ◽  
Rudolf K. F. Beran ◽  
Anna M. Pyle ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Site Directed Mutagenesis ◽  
Helical Conformation ◽  
Resonance Spectroscopy ◽  
Subgenomic Replicon ◽  
Biophysical Characterization ◽  
C Terminus ◽  
And Function

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.

The RIT1 C-terminus associates with lipid bilayers via charge complementarity

2021 ◽  
Vol 91 ◽  
pp. 107437
Author(s):  
Amy D. Migliori ◽  
Lara A. Patel ◽  
Chris Neale
Keyword(s):  
Lipid Bilayers ◽  
C Terminus

scholarly journals Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs

Cells ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 164 ◽  
Author(s):  
Mohamed Altai ◽  
Charles Leitao ◽  
Sara Rinne ◽  
Anzhelika Vorobyeva ◽  
Christina Atterby ◽  
...  
Keyword(s):  
Half Life ◽  
Molecular Design ◽  
Growth Factor Receptor ◽  
Fusion Construct ◽  
Affibody Molecules ◽  
Binding Domains ◽  
Biodistribution Studies ◽  
C Terminus ◽  
Type 3

Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.

Preparation and Characterization of a Novel Flame Retardant

2008 ◽  
Vol 47-50 ◽  
pp. 294-297 ◽  
Author(s):  
Xiu Wei Jia ◽  
Min Zhi Rong ◽  
Ming Qiu Zhang
Keyword(s):  
Flame Retardant ◽  
Sol Gel ◽  
Average Molecular Weight ◽  
Regular Structure ◽  
Gel Permeation Chromatography ◽  
Helical Conformation ◽  
Infrared Spectrometry ◽  
Resonance Spectroscopy ◽  
X Ray Diffraction

A novel flame retardant polymethylsilsesquioxane (PMSQ) was successfully obtained via combination of non-hydrolytic and hydrolytic sol-gel routes. Chemical structure of the resultant PMSQ was determined by nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectrometry and powder X-ray diffraction, respectively. All the measurements demonstrated that the product possessed regular structure with chain-to-chain width of 0.87nm and chain thickness of 0.40nm. Weight average molecular weight of PMSQ was measured to be 3.5×105 using gel permeation chromatography. Numerical simulations of the molecular structure suggested that PMSQ should exhibit cis-isotactic configuration and double helical conformation at undisturbed condition.

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