http:voh'chem'ucla'educlasses159A

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Lectures can be downloaded at the dept. voh site
at
http//voh.chem.ucla.edu/classes/159A/
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From last time - activation zone up from -40
-40
up
20
and oxyR binds in this zone to activate various
members of its regulon in response to peroxide
and related oxidants.
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References for oxyR
Zheng, M., F. Åslund, and G. Storz. (1998).
Science 2791718-1721
Kim et. al (2002), Cell, 109383-396
Choi H. et. al. Cell, 105103-113
Lee et al, Nat Struct Mol Biol. (2004) 111179-85
Tao et. al (1995) J. Bact. 177. pp. 6740-44
(later)
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A little history of peroxide challenge and oxyR
purification
Genetic pathway
Various anti-stress proteins
Peroxide treatment of cells
oxyS
oxyR induction
Some other regulators
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These forms of oxyR were used in the Storz lab
exps
4 of 6 cysteines inactivated by mutation. (others
not important for transcription - next slide)
DNA-binding domain
199
C C C C C C
208
structure
biochemistry
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Zheng, M., F. Åslund, and G. Storz. 1998.
Science 2791718-1721
4 cys gone
conclusion only 199/208 needed
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The oxidized form, when fragmented, shows a
disulfide between 199 and 208 by mass spec.
Next Is the redox system Grx1/glutathione
involved in controlling activity?
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oxyR-dependent txn. is inactivated by the GrxI
protein and reducing form of glutathione (GSH)
titration
conclusion - the oxidation state of glutathione
can determine whether oxyR will be active. (and
Grx1 acts as a catalyst for the reaction)
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Transcription starting with reduced oxyR
Reduced oxyR can be re-activated by either
peroxide or the GRX1/glutathione system. So
either direct or Grx1/glutathione oxidation
contributes to the net state.
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Model Oxidants work directly or indirectly via
glutathione oxidation. The net effect is that
oxyR monitors the cells oxidation status.
inactive
active
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Choi H. et. al. Cell, 105103-113
They went on to determine the structure of the
oxidized and reduced forms.
Oxidized form A fragment missing the
DNA-binding domain and in which 4
(non-essential) cysteines had been replaced by
site-directed mutagenesis
Reduced form Same as above except C199 has also
been replaced to prevent oxidation.
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reduced
oxidized
superpositon
disulfide
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A close-up of the region around the 2 cysteines
This arginine may enhance reactivity by
stabilizing the thiolate anion at cys-199
disulfide
Long distance between cysteines in
reduced, inactive form
Active, oxidized form
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They also found large changes in contacts between
monomers and speculated about how these could
lead to activation. But this is very far from
clear because the DNA-binding domain and the DNA
are both absent. Moreover, a subsequent paper
must be taken into account.
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Kim et. al, Cell, Vol 109, 383-396, May 2002

• They were skeptical of the oxyR model for several
  reasons including
• They say only cys199, not its partner cys208, is
  absolutely essential for activity.
• They previously found a form of oxyR in which
  only cys199 was modified,
• in this case to SNO (S-nitrosothiol)
• cys199 and cys208 are so far apart in the
  structure that it would seem to be
• difficult for them to react readily in vivo.
• it didn't fit with their prior speculation about
  the redox changes leading
• to a graded response, rather than acting via an
  on-off switch (see next slide).

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Annual Review of Genetics December 2003, Vol. 37,
pp. 91-121
Observed oxidized forms of cysteine
Only this matters to Storz
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Storz model
Kim model
Additional possible oxidants (at cys199?)
nitroso
sulfenic
glutathio
A graded response depending on redox conditions?
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Essentially, they repeated the mass-spec.analysis
using the native protein and found
"we conclude that C25 and C199 are
solvent-accessible thiols that C143 and C208
are free thiols, albeit buried in the protein
and that C180 and C259 form a disulfide, which
is accessible to reductants in the native
(aerobically purified) state Second, the finding
that residues C180 and C259 (alanines in the
crystal) participate in a disulfide bond is
supported by their very close proximity (5.9 Å).
Third, the crystallographic data places
distantly located C199 and C208 in a disulfide
(despite being 18 Å apart in the truncated
4CysAla mutant protein). We find no evidence
for a disulfide bond between C199 and C208 in
the full-length wild-type OxyR."
Uh-oh!
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Summary of their results
DNA-binding domain
Solvent accessible
199
C C C C C C
Disulfide in wt protein
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They made various oxidized forms of the solvent
accessible cysteine 199, (reduced with dtt for
comparison), and tested katG gene
activation. Conclusion all forms are active.
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Docking (right) shows that GSSG (disulfide
created by glutathione oxidation) fits perfectly
in a site near cys199. If GSSH were created by
oxidation after redox challenge it might go there
and produce cys199-S-G, which is one of their
active forms.
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CD spectra of the various forms imply a graded
degree of structure change.
And there is some evidence (see the paper) that
each form binds DNA with a unique affinity.
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(No Transcript)
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A (very recent) reply to Kim from the Storz/Ryu
groups
Nat Struct Mol Biol. 2004 Dec11(12)1179-85.
Revised protocol Use full length wt protein
(like Kim et al study). Differs in didnt use
air-oxidized protein (Kim purified as
usual). Instead reduce it fully with dtt and then
oxidize with peroxide.
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Mass spec to see which disulfides form
Reduced - all free CSH
Re-oxidized - 199-208 disulfide
Interpretation they were correct before. Other
group erred in using air-oxidized protein.
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Now look for role of cysteines in pathway of oxyR
oxidation.

• Protocol
• Modify mutants with a thiol oxidation reagent.
• 2. Detect modification via UV spectroscopy

Mutants used 4CA C208 and C199
left 4CA-C208S only C199 left 4CA-C199S
only C208 left
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Only when C199 is present alone does S-OH form
350nm known to come from sulfenic acid S-OH
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Interpretation C199 is modified to S-OH. It is
unstable and then reacts with C208 to form a
disulfide.
1
2
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no S-OH forms if rxn occurs in presence of
gluathione
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Proposal (includes rate data)
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Synthesis/cop-out in review from Georgiou (2002)
Cell 111, 607-610.
Based on the work presented suggest a plausible
model for the role of glutathione in redox
sensing. 250 words or less, please.