Cryo-EM structures of SAMD9L reveal the arrangement and coordination of multi-domains

Abstract Human sterile alpha motif domain-containing 9 (hSAMD9L) is a large (∼185 kDa) multi-domain interferon-stimulated antiviral effector with strong translation-inhibitory activity. Inherited heterozygous gain-of-function (GoF) mutations in SAMD9L directly associated with severe bone marrow failure syndromes. Using single-particle cryo-electron microscopy (cryo-EM), we determined the first structures of both full-length wild-type hSAMD9L and an N-terminal-truncated mutant at resolutions ranging from 2.8 to 3.7 Å. Both proteins exist in monomeric and dimeric states, providing clear evidence that the sterile alpha motif (SAM) and AlbA domains are not essential for dimerization. Our cryo-EM analysis reveals a tightly packed, closed architecture defined by interlocking multi-domains. We precisely mapped the extensive dimer interface mediated by Sir2-like and an oligonucleotide/oligosaccharide-binding (OB) domains. Biochemical analyses show that hSAMD9L binds double-stranded DNA in vitro but has no detectable NTP hydrolysis activity under our assay conditions. Accordingly, in our cryo-EM map we observed a clear density for a non-hydrolyzed NTP in the pocket, suggesting nucleotide binding without turnover like in STAND (Signal Transduction ATPases with Numerous Domains) proteins. Together, these results provide a structural framework for hSAMD9L and also providing key insights into its organization, domain packing and dimerization and offer a basis for understanding how GoF variants may alter hSAMD9L regulation thus impacting cell proliferation.