1. A Spring-loaded mechanism for the conformational change of Influenza Hemagglutinin
Mani Foroohar

3. 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

4. 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……

5. 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)

6. 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

7. 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

8. 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)

9. Loop Region - A Plot Twist
Classic coiled-coil heptad arrangement found…
… along with high coiling propensity…
… in the loop region?

10. Looking Deeper
Heptad repeat maintained for 88 residues from short helix through loop into long helix
Highly conserved among different strains of Influenza

11. 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

13. 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

14. 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

15. “A striking coincidence”
Loop 36 helical character
Loop 52 stability
Onset of HA membrane fusion activity
All occur in same physiologically relevant pH range

16. 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.

17. 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