Full-length influenza hemagglutinin HA2 refolds into the trimeric low-pH-induced conformation

Biochemistry. 2004 May 18;43(19):5902-11. doi: 10.1021/bi049807k.

Abstract

The influenza virus uses hemagglutinin (HA) to fuse the viral and cellular membranes. As part of an effort to study the membrane-interacting elements of HA, the fusion peptide, and the C-terminal transmembrane anchor, we have expressed in Escherichia coli the full-length HA(2) chain with maltose-binding protein fused at its N-terminus. The chimeric protein can be refolded in vitro in the presence of specific detergents to yield stable, homogeneous trimers, as determined by analytical ultracentrifugation. The trimers have the so-called "low pH" conformation-the rearranged HA(2) conformation obtained when intact HA(1)/HA(2) is induced to refold by exposure to low pH-as detected by electron microscopy and monoclonalantibody reactivity. These results provide further evidence for the notion that the neutral-pH structure of intact HA is metastable and that binding of protons lowers the kinetic barriers that prevent rearrangement to the minimum-free-energy conformation. The refolded chimeric protein described here is a suitable species for undertaking studies of how the fusion peptide inserts into membranes and assessing the nature of possible intermediates in the fusion process.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Carrier Proteins / chemical synthesis
  • Carrier Proteins / genetics
  • Carrier Proteins / ultrastructure
  • Detergents / chemistry
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli Proteins / chemical synthesis
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / ultrastructure
  • Genetic Vectors
  • Hemagglutinin Glycoproteins, Influenza Virus / biosynthesis
  • Hemagglutinin Glycoproteins, Influenza Virus / chemistry*
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics
  • Hemagglutinin Glycoproteins, Influenza Virus / ultrastructure
  • Hemagglutinins, Viral / biosynthesis
  • Hemagglutinins, Viral / chemistry*
  • Hemagglutinins, Viral / genetics
  • Hemagglutinins, Viral / ultrastructure
  • Hydrogen-Ion Concentration
  • Maltose-Binding Proteins
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Folding*
  • Recombinant Fusion Proteins / chemical synthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / ultrastructure
  • Solubility
  • Structure-Activity Relationship
  • Ultracentrifugation

Substances

  • Carrier Proteins
  • Detergents
  • Escherichia coli Proteins
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Hemagglutinins, Viral
  • Maltose-Binding Proteins
  • Recombinant Fusion Proteins
  • hemagglutinin HA-2 fusogenic peptide, Influenza virus