Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines

• Main Authors: Olivares, Adrian O. (Contributor), Kotamarthi, Hema Chandra (Contributor), Stein, Benjamin Joseph (Contributor), Sauer, Robert T. (Contributor), Baker, Tania (Contributor)
• Other Authors: Massachusetts Institute of Technology. Department of Biology (Contributor)
• Format: Article
• Language: English
• Published: National Academy of Sciences (U.S.), 2018-04-24T13:35:36Z.
• Subjects: Article
• Online Access: Get fulltext

AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use single-molecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titin[superscript I27] domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titin I27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains. Keywords:protein degradation; AAA+ proteases; directional unfolding; AAA+ motors
National Institutes of Health (U.S.) (Grant GM-101988)
National Institutes of Health (U.S.) (Grant AI-15706)