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Structure of the core ectodomain of the hepatitis C virus envelope glycoprotein 2.

Khan AG, Whidby J, Miller MT, Scarborough H, Zatorski AV, Cygan A, Price AA, Yost SA, Bohannon CD, Jacob J, Grakoui A, Marcotrigiano J - Nature (2014)

Bottom Line: Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins, whereas sheet B represents a novel fold.Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements on exposure to low pH.These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, 679 Hoes Lane West, Piscataway, New Jersey 08854, USA.

ABSTRACT
Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide. HCV infection often results in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. No vaccine is available and current therapies are effective against some, but not all, genotypes. HCV is an enveloped virus with two surface glycoproteins (E1 and E2). E2 binds to the host cell through interactions with scavenger receptor class B type I (SR-BI) and CD81, and serves as a target for neutralizing antibodies. Little is known about the molecular mechanism that mediates cell entry and membrane fusion, although E2 is predicted to be a class II viral fusion protein. Here we describe the structure of the E2 core domain in complex with an antigen-binding fragment (Fab) at 2.4 Å resolution. The E2 core has a compact, globular domain structure, consisting mostly of β-strands and random coil with two small α-helices. The strands are arranged in two, perpendicular sheets (A and B), which are held together by an extensive hydrophobic core and disulphide bonds. Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins, whereas sheet B represents a novel fold. Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements on exposure to low pH. Thus, the IgG-like fold is the only feature that E2 shares with class II membrane fusion proteins. These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.

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Hydrogen deuterium exchange and limited proteolysis of eE2(a) The percentage hydrogen deuterium exchange shown at 10, 100 and 1000 seconds time points. The secondary structure of E2 core is placed above to emphasize flexible regions. A red arrow indicates the E2 core N-terminus. Extra residues (grey) on N- and C-terminus come from the vector. Potential cleavage sites for trypsin (blue), chymotrypsin (green) and GluC (magenta) are indicated by stars. The color pattern indicates the percentage of exchange. Grey areas are the regions of no coverage. (b) Digestion of deglycosylated eE2 with chymotrypsin (left) and GluC (right) reveals a shift from the ∼35kDa untreated protein (0 min) to ∼25kDa post-digestion. Samples were taken at the indicated time points and analyzed by reducing 12% SDS-PAGE gel. Molecular weight protein standards (Std) are indicated. The bands were analyzed by N-terminal sequencing and mass spectrometry.
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Figure 3: Hydrogen deuterium exchange and limited proteolysis of eE2(a) The percentage hydrogen deuterium exchange shown at 10, 100 and 1000 seconds time points. The secondary structure of E2 core is placed above to emphasize flexible regions. A red arrow indicates the E2 core N-terminus. Extra residues (grey) on N- and C-terminus come from the vector. Potential cleavage sites for trypsin (blue), chymotrypsin (green) and GluC (magenta) are indicated by stars. The color pattern indicates the percentage of exchange. Grey areas are the regions of no coverage. (b) Digestion of deglycosylated eE2 with chymotrypsin (left) and GluC (right) reveals a shift from the ∼35kDa untreated protein (0 min) to ∼25kDa post-digestion. Samples were taken at the indicated time points and analyzed by reducing 12% SDS-PAGE gel. Molecular weight protein standards (Std) are indicated. The bands were analyzed by N-terminal sequencing and mass spectrometry.

Mentions: Solution-based studies using limited proteolysis and hydrogen deuterium exchange (HDX) demonstrated that approximately 80 amino acids on the amino terminus (384-463) from hypervariable region (HVR) 1 through HVR2 are exposed and flexible. This region includes conserved sequences implicated in binding to the cellular receptors (SR-BI and CD81) as well as several epitopes for neutralizing antibodies (Fig. 1 and Extended Data Figs. 2, 3) 7-11. Various amino-terminal deletions were produced to minimize regions of disorder while preserving an even number of cysteines, potentially allowing them to form intramolecular disulfide bonds. All constructs were screened for aggregation by non-reducing SDS-PAGE and SEC. E2 core (456-656) is soluble, monomeric, and maintains similar secondary structure content when compared with eE2 as determined by reactivity towards HCV infected patient sera (Extended Data Fig. 4a-b) and circular dichroism (data not shown). However, in contrast to eE2, CD81 binding affinity and the efficiency of inhibition of HCVcc entry was diminished for the E2 core (Extended Data Fig. 4c-e). This suggests that the amino-terminus of eE2 is critical for CD81 interaction and likely undergoes a transition from disorder to order upon binding. Alternatively, the amino-terminal region may also be ordered through interactions with other factors, e.g. E1, apolipoproteins, lipids, cellular receptors, or antibodies.


Structure of the core ectodomain of the hepatitis C virus envelope glycoprotein 2.

Khan AG, Whidby J, Miller MT, Scarborough H, Zatorski AV, Cygan A, Price AA, Yost SA, Bohannon CD, Jacob J, Grakoui A, Marcotrigiano J - Nature (2014)

Hydrogen deuterium exchange and limited proteolysis of eE2(a) The percentage hydrogen deuterium exchange shown at 10, 100 and 1000 seconds time points. The secondary structure of E2 core is placed above to emphasize flexible regions. A red arrow indicates the E2 core N-terminus. Extra residues (grey) on N- and C-terminus come from the vector. Potential cleavage sites for trypsin (blue), chymotrypsin (green) and GluC (magenta) are indicated by stars. The color pattern indicates the percentage of exchange. Grey areas are the regions of no coverage. (b) Digestion of deglycosylated eE2 with chymotrypsin (left) and GluC (right) reveals a shift from the ∼35kDa untreated protein (0 min) to ∼25kDa post-digestion. Samples were taken at the indicated time points and analyzed by reducing 12% SDS-PAGE gel. Molecular weight protein standards (Std) are indicated. The bands were analyzed by N-terminal sequencing and mass spectrometry.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126800&req=5

Figure 3: Hydrogen deuterium exchange and limited proteolysis of eE2(a) The percentage hydrogen deuterium exchange shown at 10, 100 and 1000 seconds time points. The secondary structure of E2 core is placed above to emphasize flexible regions. A red arrow indicates the E2 core N-terminus. Extra residues (grey) on N- and C-terminus come from the vector. Potential cleavage sites for trypsin (blue), chymotrypsin (green) and GluC (magenta) are indicated by stars. The color pattern indicates the percentage of exchange. Grey areas are the regions of no coverage. (b) Digestion of deglycosylated eE2 with chymotrypsin (left) and GluC (right) reveals a shift from the ∼35kDa untreated protein (0 min) to ∼25kDa post-digestion. Samples were taken at the indicated time points and analyzed by reducing 12% SDS-PAGE gel. Molecular weight protein standards (Std) are indicated. The bands were analyzed by N-terminal sequencing and mass spectrometry.
Mentions: Solution-based studies using limited proteolysis and hydrogen deuterium exchange (HDX) demonstrated that approximately 80 amino acids on the amino terminus (384-463) from hypervariable region (HVR) 1 through HVR2 are exposed and flexible. This region includes conserved sequences implicated in binding to the cellular receptors (SR-BI and CD81) as well as several epitopes for neutralizing antibodies (Fig. 1 and Extended Data Figs. 2, 3) 7-11. Various amino-terminal deletions were produced to minimize regions of disorder while preserving an even number of cysteines, potentially allowing them to form intramolecular disulfide bonds. All constructs were screened for aggregation by non-reducing SDS-PAGE and SEC. E2 core (456-656) is soluble, monomeric, and maintains similar secondary structure content when compared with eE2 as determined by reactivity towards HCV infected patient sera (Extended Data Fig. 4a-b) and circular dichroism (data not shown). However, in contrast to eE2, CD81 binding affinity and the efficiency of inhibition of HCVcc entry was diminished for the E2 core (Extended Data Fig. 4c-e). This suggests that the amino-terminus of eE2 is critical for CD81 interaction and likely undergoes a transition from disorder to order upon binding. Alternatively, the amino-terminal region may also be ordered through interactions with other factors, e.g. E1, apolipoproteins, lipids, cellular receptors, or antibodies.

Bottom Line: Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins, whereas sheet B represents a novel fold.Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements on exposure to low pH.These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, 679 Hoes Lane West, Piscataway, New Jersey 08854, USA.

ABSTRACT
Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide. HCV infection often results in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. No vaccine is available and current therapies are effective against some, but not all, genotypes. HCV is an enveloped virus with two surface glycoproteins (E1 and E2). E2 binds to the host cell through interactions with scavenger receptor class B type I (SR-BI) and CD81, and serves as a target for neutralizing antibodies. Little is known about the molecular mechanism that mediates cell entry and membrane fusion, although E2 is predicted to be a class II viral fusion protein. Here we describe the structure of the E2 core domain in complex with an antigen-binding fragment (Fab) at 2.4 Å resolution. The E2 core has a compact, globular domain structure, consisting mostly of β-strands and random coil with two small α-helices. The strands are arranged in two, perpendicular sheets (A and B), which are held together by an extensive hydrophobic core and disulphide bonds. Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins, whereas sheet B represents a novel fold. Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements on exposure to low pH. Thus, the IgG-like fold is the only feature that E2 shares with class II membrane fusion proteins. These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.

Show MeSH
Related in: MedlinePlus