SspB delivery of substrates for ClpXP proteolysis probed by the design of improved degradation tags

Abstract

The ssrA-degradation tag sequence contains contiguous binding sites for the SspB adaptor and the ClpX component of the ClpXP protease. Although SspB normally enhances ClpXP degradation of ssrA-tagged substrates, it inhibits proteolysis under conditions that prevent tethering to ClpX. By increasing the spacing between the protease and adaptor-binding determinants in the ssrA tag, substrates were obtained that displayed improved SspB-mediated binding to and degradation by ClpXP. These extended-tag substrates also showed significantly reduced conditional inhibition but bound SspB normally. Both wild-type and mutant tags showed highly dynamic SspB interactions. Together, these results strongly support delivery models in which SspB and ClpX bind concurrently to the ssrA tag, but also suggest that clashes between SspB and ClpX weaken simultaneous binding. During substrate delivery, this signal masking is overcome by tethering SspB to ClpX, which ensures local concentrations high enough to drive tag engagement. This obstruct-then-stimulate mechanism may have evolved to allow additional levels of regulation and could be a common trait of adaptor-mediated protein degradation.energy-dependent degradation ͉ degradation specificity ͉ degradation signals ͉ compartmental protease ͉ adaptor proteins

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

“…As an alternative approach for improving the apparent degradation efficiency of the negative control and the reporter molecules in general, we tried using an engineered ssrA tag containing an extra pair of asparagine and tyrosine residues (boldface) (AANDENYNYALAA), which has been reported to improve ClpXP-mediated degradation of ssrAtagged proteins (21). When this modified tag was used, we actually observed an improved resolution between fully induced positive (DH5␣/pMal-TEV2/pGFP-subG-ssrA NY ) and negative (DH5␣/pMal-TEV2/pGFP-ssrA NY ) cells, compared to cells that instead employed the normal ssrA tag in the reporter constructs.…”

“…The amplicon was then digested with SacI and HindIII and ligated into the SacI/HindIII-digested backbone of pBAD33 (19), yielding pGFP-ssrA. A very similar plasmid, pGFPssrA NY , that instead encodes a modified ssrA tag containing an additional pair of asparagine and tyrosine residues (in boldface) (AANDENYNYALAA), which improves ClpXP-mediated degradation efficiency (21), was also constructed. This was done by a QuikChange site-directed mutagenesis reaction (Stratagene) with primers GEKO14 and GEKO15 on the template pGFP-ssrA.…”

In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

“…However, ClpXP uses the SspB adaptor protein bound to a secondary site of the SsrA tag to enhance recognition of the tagged protein (12,19). SspB binding to the SsrA tag inhibits the ClpAP system, pointing to structural differences between these two protease systems.…”

Degradation of SsrA-tagged proteins in streptococci

“…In vitro degradation assays were performed as described previously (Hersch et al, 2004;Kim et al, 2000). Briefly, 0.3 mM His-ClpX, 0.3 mM His-ClpE, 0.3 mM His-ClpC, 0.1 mM MecA, 0.8 mM ClpP-His and/or 0.1 mM substrate GFPs were added to a 100 ml reaction system.…”

“…To verify the potential of other SspB-like tethering roles in S. mutans, we replaced the last three residues, VAA, in the S. mutans SsrA tag to LGG. A four-residue linker, SENY, was added before the last four residues of the SsrA tag to enhance the tethering role, as reported previously (Hersch et al, 2004;McGinness et al, 2006McGinness et al, , 2007. The modified SsrA tags, SENY-AVAA, SENY-ALGG and AVAA, were then added to the C terminus of GFP; the resulting GFP derivatives were named GFPssrA, GFPssrA*, GFPavaa, GFPalgg and GFPavaaNL.…”