title: Global fold and backbone dynamics of the hepatitis C virus E2 glycoprotein transmembrane domain determined by NMR.
abstract: E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM-TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717-726 and 732-746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3ns. The positioning of the helix-linker-helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization.
authors.affiliation: Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel.
authors.forename: Hila
authors.initials: H
authors.lastname: Shalom-Elazari
authors.affiliation: Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel.
authors.forename: Hadas
authors.initials: H
authors.lastname: Zazrin-Greenspon
authors.affiliation: Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel.
authors.forename: Hadassa
authors.initials: H
authors.lastname: Shaked
authors.affiliation: Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel. Electronic address: Jordan.Chill@biu.ac.il.
authors.forename: Jordan H
authors.initials: JH
authors.lastname: Chill
keywords: Envelope glycoproteins; Hepatitis C virus; Membrane-associated proteins; Nuclear magnetic resonance; Protein structure; Transmembrane helix