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 ClpPBinding 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.

Formed 2 mutants Mutant mutated to generate GFP – D 2 A 5 DLAA Mutant mutated to glycines – GFP – G 8 LAA Is L-A-A sequence enough for ClpX recognition?

Mutant 1 caused slight Km increase due to cumulative minor effects. Glycine-rich Mutant 2 inhibited ClpX recognition due to flexibility.

The tri-peptide LAA sequence is sufficient in most cases (but not all) for ClpX recognition. Conclusion

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?

Compared normal  - carboxyl group to mutant terminal carboxamide (ssrA -CONH2) group. Testing for  -carboxyl group recognition

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.

C-terminal tripeptide of ssrA tag is highly conserved across many bacterial species. N-terminal portions of ssrA also highly conserved Conservation of Clpx and SspB

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