The promiscuous metalloprotease SPRTN is the key enzyme for proteolytic repair of DNA-protein crosslinks (DPCs). To prevent uncontrolled SPRTN activity, its activation must be tightly regulated. Using NMR spectroscopy and in vitro reconstitution, we elucidate the molecular basis of SPRTN's activation by DNA and ubiquitin. We identify an autoinhibitory mechanism governed by intramolecular electrostatic interactions between a negatively charged linker helix and SPRTN's positively charged DNA-binding domains. DNA relieves this autoinhibition by competitively displacing the linker from the DNA-binding domains, thereby inducing a conformational shift to an open, catalytically active state. This open state enables ubiquitin binding to SPRTN's protease domain, further stabilizing the active conformation. Disruption of the interaction between the autoinhibitory linker and the DNA-binding domains locks SPRTN in a constitutively open state, resulting in enhanced protease activity. Collectively, our data reveal how DNA and ubiquitin cooperate to convert SPRTN from an autoinhibited conformation into its active state.
Kang, H.-S., Dürauer, S., Renz, C., Machida, Y., Ulrich, H. D., Machida, Y. J., Sattler, M., Stingele, J.
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