Transcript The unfolded protein response signals through high

The unfolded protein response
signals through high-order
assembly of Ire1
Alexei V. Korennykh1,3, Pascal F. Egea1, Andrei A. Korostelev4, Janet
Finer-Moore1, Chao Zhang2,3, Kevan M. Shokat2,3, Robert M. Stroud1 &
Peter Walter1,3
Alysia Birkholz
1.15.2010
• ~33% of proteins fold in the ER
• Need a mechanism to detect misfolded
proteins
– UPR
– Ire1 is the crucial factor
• Has been shown to dimerize
• Organized into UPR induced foci
• RNase
– Trans-autophosphorylation
– Binding of ADP
Ire1
“This pathway is really a sort
of duckbilled platypus. There
have been so many
surprises.”
- Feroz Papa UCSF
• Inositol-requiring protein-1
• ER-membrane-resident kinase/RNase
• a non-spliceosomal mRNA splicing reaction
– In yeast HAC1 mRNA
• HAC1p proteins are then translated
– In metazoans, Xbp1 mRNA
• Xbp1 proteins are then translated
– These proteins adjust protein folding
In stressed cells, IRE-1 binds
to unfolded proteins,
dimerizes and transautophosphorylates in
kinase domain
This activates the RNase
domain which splices XBP1
or HAC1
The spliced form of these
genes leads to activation of
the UPR
BIOC/MCB 568 -- University of Arizona
Activation of Ire1 by self-association.
Oligomerization
has been
suggested to
promote the
activation the
kinase and
RNase domains
Binding sites of Ire1 in
metazoans (right) and yeast
(left)
Aim
• Authors didn’t like the proposed model that
had the activation loops too far away for
trans-autophosphorylation
– Previous model had no linker component
– A different Ire1 dimer is needed for the transautophosphorylation
RNase activity assay:
Construct cleaved is
labeled with P32 and is
derived from Xbp1 mRNA
Linker controls the oligomerization
Oligomerization
is inhibited by
increasing salt
concentrationsrepressing RNase
activity
Ire1KR32 shows fold increase in speed
of cleavage
Structure of the oligomer
• Difficult to crystalize
• ADP association wasn’t working
• Tried using kinase inhibitors – insert into ADP
binding site
Kinase inhibitors
actually activated
the RNase activity
Resolution of crystals
Resolution (Å)
Structural features observable in a good map
5.5
Overall shape of the molecule. Helices as rods of strong intensity.
3.5
The main chain (usually with some ambiguities).
3.0
The side chains partly resolved.
2.5
Side chains well resolved. The plane of the peptide bond resolved.
Atoms located to about ± 0.4 Å.
1.5
Atoms located to about ± 0.1 Å (the limit of protein crystallography
as of 1985--it's a little bit lower now)
0.77
Bond lengths in small crystals measured to 0.005 Å.
• Initially got a Ire1- APY29
complex of 3.9 A
• When they deleted 28 amino
acids forming a loop, got a
resolution of 3.2 A
Electron density map for
APY29 bound to
IRE1KR32Δ28
3 possible hydrogen
bonds formed
Although APY29 fits in
the ADP site reasonably
well, it doesn’t have
the divalent Mg 2+
RNase activity of APY29
doesn’t change with
addition of EDTA
14 Ire molecules formed a crystal
lattice with back to back dimers
Double Helix
Why is this model attractive?
• Kinase dimers are side by side, making transautophosphorylation more logical
• Kinase inactivating mutant caused no
phosphorylation to occur, indicating
phosphorylation activity is regulated through
self
• Phosphorylation sites are on activation loops
which face IF3c
– IF3c proposed to transfer phosphates
Do all three interfaces control RNase activity?
Four monomers
combined
Single
monomer
Contacts
within the
different
interfaces
The mechanism
• Initially showed that the IreKR32 seemed to
show preference for double stem loop mRNA
• To look at this, generated a 354 ntd RNA with
a single stem loop, and a 58 ntd RNA with a
double stem loop
The mechanism
Ire1 has no
preference for
single or
double loops
Localization of Helix loop helix
near IF2 interface (dimerization
interface) – may control RNase
activity
Cavity is created that is
characteristic for substrate
binding pockets of enzymes
Model
Conclusions
• Old model is incomplete
• Ire1 kinase, Rnase, and partial linker forms a
supermolecular structure
• Co-factor binding and phosphorylation
enhance self-association of Ire1 but neither is
required
• Linkers are necessary for oligomerization
Future work
• Although the authors used an increased
structure for crystallization, we are still
missing the full structure
• Need to have evidence that these
supermolecular structures are actually present
in in vitro/ in vivo models
?