Nucleotide-dependent substrate handoff from the SspB adaptor to the AAA+ ClpXP protease.
The SspB adaptor enhances ClpXP degradation by binding the ssrA degradation tag of substrates and the AAA+ ClpX unfoldase. To probe the mechanism of substrate delivery, we engineered a disulfide bond between the ssrA tag and SspB and demonstrated otherwise normal interactions by solving the crystal structure. Although the covalent link prevents adaptor.substrate dissociation, ClpXP degraded GFP-ssrA that was disulfide bonded to the adaptor. Thus, crosslinked substrate must be handed directly from SspB to ClpX. The ssrA tag in the covalent adaptor complex interacted with ClpX.ATPgammaS but not ClpX.ADP, suggesting that handoff occurs in the ATP bound enzyme. By contrast, SspB alone bound ClpX in both nucleotide states. Similar handoff mechanisms will undoubtedly be used by many AAA+ adaptors and enzymes, allowing assembly of delivery complexes in either nucleotide state, engagement of the recognition tag in the ATP state, and application of an unfolding force to the attached protein following hydrolysis.
Bolon
Daniel N
DN
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA.
Grant
Robert A
RA
Baker
Tania A
TA
Sauer
Robert T
RT
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D002352:Carrier Proteins; D004220:Disulfides / Q000737:chemistry*; D049071:Endopeptidase Clp / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006193:Haemophilus influenzae / Q000201:enzymology; D006868:Hydrolysis; D008958:Models, Molecular; D017510:Protein Folding; D021122:Protein Subunits; D012329:RNA, Bacterial / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D002352:Carrier Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; C477700:sspB protein, E coli; C096222:tmRNA; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases