Abstract
SspB is a dimeric adaptor protein that increases the rate at which ssrA-tagged substrates are degraded by tethering them to the ClpXP protease. Each SspB subunit consists of a folded domain that forms the dimer interface and a flexible C-terminal tail. Ternary delivery complexes are stabilized by three sets of tethering interactions. The C-terminal XB peptide of each SspB subunit binds ClpX, the body of SspB binds one part of the ssrA-tag sequence, and ClpX binds another part of the tag. To test the functional importance of these tethering interactions, we engineered monomeric SspB variants and dimeric variants with different length linkers between the SspB body and the XB peptide and employed substrates with degradation tags that bind ClpX weakly and/or contain extensions between the binding sites for SspB and ClpX. We find that monomeric SspB variants can enhance ClpXP degradation of a subset of substrates, that doubling the number of tethering interactions stimulates degradation via changes in Km and Vmax, and that major alterations in the length of the 48-residue SspB linker cause only small changes in the efficiency of substrate delivery. These results indicate that the properties of the degradation tag and the number of SspB.ClpX tethering interactions are the major factors that determine the extent to which the substrate and ClpX are engaged in ternary delivery complexes.
Highlights
Escherichia coli ClpXP is an ATP-dependent protease [3]
The results presented here show that a monomeric, singletailed SspB variant stimulates ClpXP degradation of substrates bearing extended versions of the ssrA and DAS degradation tags
This finding demonstrates that there is no intrinsic requirement for bivalent tethering or a functional requirement for a second SspB subunit in adaptor-mediated substrate delivery to the ClpXP protease
Summary
Monomeric SspB was generated by introducing eight different mutations (see below) into a plasmid-borne gene for E. coli SspB Another monomeric SspB with an N-terminal His tag was constructed in pET24d for biophysical studies. To generate a variant with the longer tail of SspBLL91, a SacII site was introduced by silent mutation of the codons for residues 153 and 154 in the parent plasmid, the resulting plasmid was digested with SacII and HindIII, and a SacII-HindIII PCR fragment encoding residues 113–165 of the E. coli SspB tail was inserted by ligation. Plasmids encoding E. coli ClpX, E. coli ClpP-His, and a His6tagged variant of chimeric SspB with the H. influenzae substrate binding domain and the E. coli tail have been described [11, 20, 21]. Fluorescence anisotropy values were measured for 2 min and averaged
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