Overlapping recognition determinants within the
ssrA degradation tag allow modulation of
proteolysis
Flynn, Levchenko, Seidl, Wickner, Sauer, Baker
Presented by Alice Crane and Lindsey Wu
How do ClpX and ClpA interact differently
with SspB?
Definitions
ssrA
peptide sequence added to proteins targeted for
degradation
ClpX and ClpA
Proteins that promote ATP dependant degradation, aid in
unfolding of protein
ClpP
Protease that contains active site for degradation of
damaged proteins
Definitions continued
ClpXP and ClpAP
Complexes formed by these proteins that carry out
degradation
SspB
Regulator of substrate recognition
- enhances ClpX recognition
- inhibits ClpA recognition
Proteolytic chamber of ClpP
Binding to ssrA tag
Goal of Experiment
To determine the sequence information in the 11 a.a
ssrA degradation tag required for recognition by ClpX,
ClpA, and SspB
Mutant Derivation
Non alanine residues  Alanine (A)
Alanine residues  Aspartic Acid (D)
GFP Mutants
GFP cloned into a vector and mutant ssrA tags ligated into
vector.
Allows for loss of fluorescence to be measured – basis for
for degradation assays.
Loss in fluorescence = Increase in degradation
SsrA Tag ClpX Recognition
Goal: To determine which residues are critical for ClpX
recognition by testing 12 GFP-ssrA mutants.
Only those with substitutions at tag positions 9, 10 and
11 caused significant increase in Km for ClpXP
degradation, though 9 was less critical.
Is L-A-A sequence enough for ClpX recognition?
Formed 2 mutants
Mutant 1
1-8 mutated to generate GFP – D2A5DLAA
Mutant 2
1-8 mutated to glycines – GFP – G8LAA
Mutant 1 caused slight Km
increase due to cumulative
minor effects.
Glycine-rich Mutant 2
inhibited ClpX recognition
due to flexibility.
Conclusion
The tri-peptide LAA sequence is sufficient in most
cases (but not all) for ClpX recognition.
SsrA Tag ClpA Recognition
Goal: To determine which residues are critical for ClpA
recognition by using 12 GFP-ssrA mutants
ClpA relies on a different set of residues than ClpX.
ClpA recognizes sequences at N terminus and C terminus
Mutation of C terminal Alanine had no effect on
degradation.
So, is the free -carboxyl group necessary for recognition?
Testing for -carboxyl group recognition
Compared normal - carboxyl group to mutant terminal
carboxamide (ssrA -CONH2) group.
Conclusions
ClpA recognizes ssrA-like signals in any exposed
region of a protein.
(Previous studies show that -carboxyl group is
important for ClpX recognition of ssrA)
SsrA Tag SspB Recognition
Goal: To determine which residues are critical for SspB
recognition
Created peptide library
Each residue mutated to each other 19 aa’s, while
keeping other 10 residues unchanged.
(Total of 220 mutants)
Each spot corresponds to
1 mutant sequence.
Bound SspB was detected
with anti-SspB antibodies.
SspB recognition is
dependent on residues
1,2,3,4, and 7.
SspB and ClpX are recognition dependent on different
positions on ssrA tag.
SspB and ClpA interact with some of the same residues.
Conclusion
SspB enhances ClpX recognition, but inhibits ClpA.
Dual Recognition by SspB and ClpX
Goal
To determine if binding of SspB to ssrA tag is enough
for ClpX recognition or is independent recognition of
ClpX required.
Monitored degradation with
SspB of 3 mutants (L9A,
A10D, A11D).
All are ClpX recognition
defective.
Mutants A10D and A11D were
not degraded by ClpX.
Mutant L9A with SspB was
degraded, but not as
efficiently as wild type with
SspB.
Conclusions
SspB binding cannot bypass requirement of ClpX
recognition for residues 10 and 11.
SspB can compensate for decreased interaction with
ClpX and a mutation at residue 9.
SspB regulated degradation requires sets of binding
determinants for both ClpX and SspB.
SspB Inhibition of ClpA
Goal
To test the assumption that ClpA and SspB binding is
mutually exclusive.
Compared wild type to mutant
(N3A) defective in SspB
recognition.
Wild type ClpA recognition
completely inhibited
Mutant ClpA recognition not
inhibited SspB presence.
Conclusions
Binding is mutually exclusive.
Specific interaction of SspB to ssrA tag required to inhibit
ClpA recognition.
Conservation of Clpx and SspB
C-terminal tripeptide of ssrA tag is
highly conserved across many
bacterial species.
N-terminal portions of ssrA also
highly conserved
Conclusion
Suggests that these bacteria also have a SspB-like regulator
or that these are regions mediate interactions with other
proteases.
What is the biological explanation for the inhibition of ClpA?
Possible Explanations
ClpAP, but not ClpXP, degrades unfolded proteins without
target signals.
- important during heat shock or environmental stress
In times of stress, up-regulation of SspB can direct ssrA
tagged substrates to ClpXP, leaving ClpAP free to degraded
unfolded substrates.
Works Cited
Clark, Adrien K. “ATP-dependent Clp Proteases in Photosynthetic OrganismsÐ
A Cut Above the Rest!” (1999) Annals of Botany 83: 593±599
Hersch, Greg L. et al. “SspB delivery of substrates for ClpXP proteolysis
probed by the design of improved degradation tags.” (2004) Proc. Natl. Acad.
Sci. 101: 12136–12141