SspB delivery of substrates for ClpXP proteolysis probed by the design of improved degradation tags.
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.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Greg L
GL
Hersch
Tania A
TA
Baker
Robert T
RT
Sauer
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D021122:Protein Subunits; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D021122:Protein Subunits; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; C477700:sspB protein, E coli; C096222:tmRNA; D049452:Green Fluorescent Proteins; D012697:Serine Endopeptidases; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases