title: Architecture of the hepatitis C virus E1 glycoprotein transmembrane domain studied by NMR.
abstract: Oligomerization of hepatitis C viral envelope proteins E1 and E2 is essential to virus fusion and assembly. Although interactions within the transmembrane (TM) domains of these glycoproteins have proven contributions to the E1/E2 heterodimerization process and consequent infectivity, there is little structural information on this entry mechanism. Here, as a first step towards our long-term goal of understanding the interaction between E1 and E2 TM-domains, we have expressed, purified and characterized E1-TM using structural biomolecular NMR methods. An MBP-fusion expression system yielded sufficient quantities of pure E1-TM, which was solubilized in two membrane-mimicking environments, SDS- and LPPG-micelles, affording samples amenable to NMR studies. Triple resonance assignment experiments and relaxation measurements provided information on the secondary structure and global fold of E1-TM in these environments. In SDS micelles E1-TM adopts a helical conformation, with helical stretches at residues 354-363 and 371-379 separated by a more flexible segment of residues 364-370. In LPPG micelles a helical conformation was observed for residues 354-377 with greater flexibility in the 366-367 dyad, suggesting LPPG provides a more native environment for the peptide. Replacement of key positively charged residue K370 with an alanine did not affect the secondary structure of E1-TM but did change the relative positioning within the micelle of the two helices. These results lay the foundation for structure determination of E1-TM and a molecular understanding of how E1-TM flexibility enhances its interaction with E2-TM during heterodimerization and membrane fusion.
authors.affiliation: Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel.
authors.forename: Hadas
authors.initials: H
authors.lastname: Zazrin
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
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D006017:Glycolipids / Q000737:chemistry / Q000378:metabolism; D007295:Inositol Phosphates / Q000737:chemistry / Q000378:metabolism; D008823:Micelles; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C087035:E1 protein, Hepatitis C virus; D006017:Glycolipids; D007295:Inositol Phosphates; C111513:LPPG glycoinositolphosphoceramide; D008823:Micelles; D010446:Peptide Fragments; D014759:Viral Envelope Proteins
keywords: 1,4-dithio-D-threitol; 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol); 4,4-dimethyl-4-silapentane-1-sulfonic acid; CD; CPMG; Carr–Purcell–Meiboom–Gil (pulse-train); DHPC; DPC; DSS; DTT; Envelope glycoproteins; HCV; HSQC; Hepatitis C virus; IPTG; LPPG; MBP; MP; Membrane-associated proteins; NMR; Nuclear magnetic resonance; Protein structure; SDS; TEV; TM; Transmembrane helix; circular dichroism; dihexanoylphosphatidylcholine; dodecyl-phosphocholine; hepatitis C virus; heteronuclear single-quantum coherence; isopropyl β-D-1-thiogalactopyranoside; maltose-binding protein; membrane-embedded protein; nuclear magnetic resonance; sodium dodecylsulphate; tobacco etch virus (protease); transmembrane domain