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 [A]. 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.