a spring loaded mechanism for the conformational change of influenza hemagglutinin l.
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A Spring-loaded mechanism for the conformational change of Influenza Hemagglutinin. Mani Foroohar. The Story So Far…. Influenza Hemagglutinin (HA) undergoes conformational change that induces fusion Sequence with high propensity to form coiled-coil identified

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Presentation Transcript
the story so far
The Story So Far….
  • Influenza Hemagglutinin (HA) undergoes conformational change that induces fusion
  • Sequence with high propensity to form coiled-coil identified
  • Localized to loop region in native form
  • Findings suggest a model for fusogenic conformational change in HA
what s my motivation
What’s my motivation?
  • Membrane fusion critical in diverse processes
  • Process is slow in the absence of specific proteins
  • Potential to provide drug targets in the "future”
  • Provides insight into mechanisms underlying changes in protein conformation as a response to changing environmental conditions
  • I’d like not to fail Bioc 230……
scene of the crime
Scene of the Crime
  • Influenza selected for use in this study for its well-characterized cell fusion
  • Viral binding to sialic acid (activity of HA in native conformation) precedes fusion
  • Bound virion internalized by receptor-mediated endocytosis
  • drop in pH leads to envelope fusion with mature endosome (Wiley and Skehel, 1987; Stegmann and Helenius 1993)
  • Trimeric glycoprotein HA promotes membrane fusion
  • At low pH, HA is sufficient for fusion in vivo and in vitro (Whiter et al., 1982)
getting there is half the battle
Getting there is half the battle
  • HA synthesized as precursor HA0
  • HA0 processed proteolytically to disulfide-bonded HA1 and HA2
  • processing necessary for viral infectivity (Lazarowitz and Choppin, 1975)
  • Native HA binds sialic acid, but lacks fusion activity
  • Mildly acidic conditions in mature endosome (pH 5) induce necessary conformational change
ha1 knocking on the door
HA1 - Knocking on the Door
  • Exoplasmic domain
  • Sialic acid binding activity
  • Distal tip 135 angstroms from viral surface
  • Assembles atop stem formed of HA2 coils
ha2 breaking and entering
HA2 - Breaking and Entering
  • Transmembrane subunit
  • Short a helix connected to long a helix by loop
  • Long helices form three stranded coiled-coil
  • Short helices displayed external to coil
  • Highly conserved “fusion peptide” (aa 1- 25)
loop region a plot twist
Loop Region - A Plot Twist
  • Classic coiled-coil heptad arrangement found…
  • … along with high coiling propensity…
  • … in the loop region?
looking deeper
Looking Deeper
  • Heptad repeat maintained for 88 residues from short helix through loop into long helix
  • Highly conserved among different strains of Influenza
hypothesis
Hypothesis:
  • HA2 folds into one long three stranded coiled-coil in its fusogenic state
  • Existing coil is extended to include short external helix and loop region
  • 80 angstrom coiled coil extends to 135 angstroms
  • Fusion peptide displaced 100 angstroms toward target membrane to promote fusion
loop 36
Loop 36
  • Sequence exhibits heptad repeat pattern
  • Random coil at pH 7, coiled coil at pH 4.8
  • Unfolding transition seen at pH 4.8, but not pH 7
  • Molecular mass (slope of graph) equal to trimer at pH 4.8, monomer at pH 7
  • Helix content highest in pH 4 to pH 5 range
loop 52
Loop 52
  • Heptad repeat
  • Characteristic a-helical spectra at pH 4.8 and 7
  • Avg. molecular mass consistent with trimer at pH 4.8 and 7
  • Exhibits thermal unfolding transition at pH 7 and 4.8
a striking coincidence
“A striking coincidence”
  • Loop 36 helical character
  • Loop 52 stability
  • Onset of HA membrane fusion activity
  • All occur in same physiologically relevant pH range
additional evidence
Additional Evidence
  • Short and long helices and loop of HA2 have shown resistance to degradation in proteolysis experiments
  • Loop region (which should be vulnerable to proteolysis) displays resistance in the fusogenic state, in keeping with the suggestion that it becomes part of a coiled coil
  • Folding of HA0 into native state is ATP-dependant, suggesting that it may be less stable than the extended coiled-coil.
conclusions
Conclusions
  • Formation of extended coiled coil supported by aa sequence analysis and evidence of pH dependant folding
  • Previous proteolysis studies by other investigators provide additional support, but none specifically testing the “spring-loading” hypothesis are available.
  • Findings of this paper may have relevance to other fusion events, such as release of gp120 from gp41 in HIV