Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of an AAA+ degradation machine.
Proteolytic machines powered by ATP hydrolysis bind proteins with specific peptide tags, denature these substrates, and translocate them into a sequestered compartment for degradation. To determine how ATP is used during individual reaction steps, we assayed ClpXP degradation of ssrA-tagged titin variants with different stabilities in native and denatured forms. The rate of ATP turnover was 4-fold slower during denaturation than translocation. Importantly, this reduced turnover rate was constant during denaturation of native variants with different stabilities, but total ATP consumption increased with substrate stability, suggesting an iterative application of a uniform, mechanical unfolding force. Destabilization of substrate structure near the degradation tag accelerated degradation and dramatically reduced ATP consumption, revealing an important role for local protein stability in resisting denaturation. The ability to denature more stable proteins simply by using more ATP endows ClpX with a robust unfolding activity required for its biological roles in degradation and complex disassembly.
Kenniston
Jon A
JA
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Baker
Tania A
TA
Fernandez
Julio M
JM
Sauer
Robert T
RT
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D064211:Connectin; D049071:Endopeptidase Clp; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000502:physiology; D012697:Serine Endopeptidases / Q000378:metabolism*
D001426:Bacterial Proteins; D064211:Connectin; D009124:Muscle Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases