Structural and Biochemical Analysis of Human Schlafen5 Reveals the Molecular Basis of Its Anti-Tumor Function

Background: Schlafen5 (SLFN5) is an IFN-inducible member of the SLFN protein family involved in immune regulation, viral inhibition and tumor control. Recently, SLFN5 was suggested to inhibit the invasion of many kinds of malignant cells, and its expression positively correlates with the overall survival of relevant patients. However, the functional mechanism of SLFN5 remains elusive. Here, we aim to investigate the biochemical properties and anti-tumor mechanism of SLFN5. Methods: We expressed, purified and crystallized the N-terminal domain of human SLFN5 (hSLFN5-N) and solved its structure by molecular replacement. We examined the binding and digestion ability of hSLFN5-N to several kinds of nucleic acid substrates, including DNA, RNA, tRNA and rRNA via electrophoretic mobility shift assay (EMSA) and cleavage assay in vitro. Structure-based mutation screening and cancer-associated hot-spot single-point mutations in hSLFN5 were analyzed to reveal the critical amino acid sites that influence the structure, biochemical and physiological function of the protein. Results: Structurally, hSLFN5-N is characterized by a pseudo-dimeric domain organization with a flexible central valley that is narrower than that of rat (r)SLFN13-N. Unlike rSLFN13-N, hSLFN5-N is incapable of cleaving tRNA and rRNA, but it is able to bind DNA and cleave specific RNA substrates. Site-mutation analysis suggested that positively charged residues surrounding the central valley determine the DNA and RNA affinity of hSLFN5. Structure-based analysis revealed that cancer-associated mutations tend to severely affect the folding stability and function of SLFN5 by disrupting intra-/inter-molecular interactions, incurring unfavorable alterations in local conformation, or altering potential post-translational modifications (PTMs) of the wild-type protein. Besides, SLFN5 had no DNA sequence selectivity in A375 cells, which indicated that its anti-migration activity may mainly depend on its RNA manipulation ability rather than on its DNA affinity as a transcription factor. Conclusions: SLFN5 is a DNA- and RNA-binding protein with endonuclease activity on specific RNA substrates, which determines the role of SLFN5 as a tumor suppressor. Single-point mutations may disrupt the structural stability, RNA manipulation capacity and thus the tumor suppressor function of SLFN5.