DNA-induced conformational changes in SPRTN relieve its auto-inhibitory effect on protease activity.

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• The DNA-dependent metalloprotease SPRTN is vital for resolving DNA-protein crosslinks (DPCs), preventing genome instability, aging, and cancer.
• DNA and ubiquitin chains activate SPRTN, but the precise mechanisms remain unclear.

Purpose of the Study:

• To elucidate the activation mechanism of SPRTN by DNA and ubiquitin.
• To characterize the enzymatic activity and regulation of SPRTN fragments.

Main Methods:

• Development of a sensitive multi-turnover FRET assay for real-time SPRTN proteolysis monitoring.
• Site-trapping experiments to capture intermediate conformations.

Main Results:

• Auto-cleaved SPRTN N-terminal fragment (MPD, ZBD, BR) is stable, active, and ubiquitin-dependent.
• Metalloprotease domain (MPD) alone has basal activity; ZBD and MPD regulate each other's activity and DNA binding.
• DNA binding, mediated by BR, relieves ZBD-induced autoinhibition of MPD, inducing an open conformation.

Conclusions:

• SPRTN auto-cleavage does not inactivate the enzyme; instead, it primes it for activation.
• DNA binding induces critical conformational changes in SPRTN, facilitating ubiquitin activation and efficient DPC proteolysis.
• This study reveals a novel regulatory mechanism for SPRTN, essential for maintaining genome integrity.