Structural and functional characterization of human SLFN14.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• The Schlafen (SLFN) protein family are DNA/RNA processing enzymes involved in immunological and anti-tumor processes.
• SLFN14, a member of this family, exhibits antiviral activity and is linked to an inherited bleeding disorder.

Purpose of the Study:

• To present the cryo-electron microscopy structure of full-length human SLFN14.
• To elucidate the structural and biochemical features of SLFN14, including its enzymatic activities and potential cellular conformations.

Main Methods:

• Cryo-electron microscopy (cryo-EM) for structural determination.
• Biochemical assays to assess RNase and helicase activity.
• Live-cell fluorescence resonance energy transfer (FRET) assays and AlphaFold2 analysis for conformational studies.

Main Results:

• The structure of full-length human SLFN14 was determined, revealing RNase, SWADL, and helicase domains.
• SLFN14 demonstrated potent RNase activity, with specific charged patches in the RNase domain crucial for oligonucleotide binding.
• SLFN14 lacked helicase activity due to an inability to bind ATP and the absence of positive charges in its DNA-binding site.
• Structural comparisons showed SLFN14 is similar to SLFN11 but differs from SLFN5 in helicase domain orientation.
• FRET assays and AlphaFold2 suggested SLFN14 may adopt multiple conformations in cells.

Conclusions:

• Detailed structural and biochemical insights into SLFN14 were provided.
• The findings expand the understanding of the functional diversity within the SLFN protein family.
• The study highlights SLFN14's role in RNA processing and its potential implications in disease.