Transcript A Spring-loaded mechanism for the conformational change of

A Spring-loaded mechanism for the
conformational change of Influenza
Hemagglutinin
Mani Foroohar
The Story So Far….
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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?
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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
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Influenza selected for use in this study for its wellcharacterized 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
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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
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Exoplasmic domain
Sialic acid binding
activity
Distal tip 135 angstroms
from viral surface
Assembles atop stem
formed of HA2 coils
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
HA2 - Breaking and Entering
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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)
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Loop Region - A Plot Twist
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Classic coiled-coil
heptad arrangement
found…
… along with high
coiling propensity…
… in the loop region?
Looking Deeper
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Heptad repeat maintained for 88 residues from short
helix through loop into long helix
Highly conserved among different strains of Influenza
Hypothesis:
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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
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Loop 36
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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
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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”
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Loop 36 helical character
Loop 52 stability
Onset of HA membrane fusion activity
All occur in same physiologically relevant pH range
Additional Evidence
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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
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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