Altered tethering of the SspB adaptor to the ClpXP protease causes changes in substrate delivery.
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.
McGinness
Kathleen E
KE
Department of Biology and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Bolon
Daniel N
DN
Kaganovich
Mark
M
Baker
Tania A
TA
Sauer
Robert T
RT
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006193:Haemophilus influenzae / Q000201:enzymology; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D013379:Substrate Specificity
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D047908:Intracellular Signaling Peptides and Proteins; C482539:SspB specificity factor, Hemophilus influenzae; C477700:sspB protein, E coli; D049071:Endopeptidase Clp