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abstracts
SAMHD1, a deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase), prevents the infection of blood cells by retroviruses, including HIV, by depleting the cellular dNTP pool available for viral reverse transcription. SAMHD1 is a major regulator of cellular dNTP levels in mammalian cells. Mutations in SAMHD1 are associated with the autoimmune condition Aicardi Goutières Syndrome (AGS), whose clinical manifestations resemble congenital viral infection. The catalytic activity of SAMHD1 is regulated by allosteric binding of dGTP, which enables SAMHD1 monomers/dimers to assemble into the catalytically active tetrameric form. We have determined the crystal structure of the tetrameric human SAMHD1-dGTP complex. The structure reveals an elegant allosteric mechanism of activation via dGTP-induced assembly of the tetrameric complex from two inactive dimers. Intriguingly, GTP can also activate SAMHD1, and our data further show the binding promiscuity of other dNTPs at the allosteric site. These findings suggest a regulation system that may have a profound effect on the balancing of cellular dNTP pools. These results provide the basis for a mechanistic understanding of SAMHD1 function in HIV restriction, the pathogenesis of AGS, and regulation of cellular dNTP levels.
abstracts
RNase III represents a family of dsRNA-specific endoribonucleases required for RNA maturation and gene regulation. Bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer are representative members of the family. The bacterial enzyme possesses a single RNase III domain (RIIID) followed by a dsRNA-binding domain (dsRBD); Rnt1p is defined by the presence of an N-terminal domain (NTD), a RIIID, and a dsRBD; Drosha contains N-terminal P-rich and RS-rich domains followed by two RIIIDs and a dsRBD; and Dicer possesses N-terminal helicase, DUF283, and PAZ domains followed by two RIIIDs and a dsRBD. It is the N-terminal extension beyond the RIIID that distinguishes eukaryotic RNase IIIs from the bacterial enzyme. My lab has been studying the structure and mechanism of RNase III enzymes since 1996. We have reported a total of eleven crystal structures of bacterial RNase III in complex with dsRNA at various catalytic stages of the enzyme, including the first structure of a catalytically meaningful RNase III-RNA complex (Gan et al., Cell, 124:355-366, 2006), and thereby well characterized the mechanism of action for the bacterial enzyme (Court et al., Annu Rev Genet, 47:405-431, 2013). We have also determined the crystal structure of yeast Rnt1p post-cleavage complex, the first structure of a eukaryotic RNase III complexed with RNA in a catalytically meaningful manner (Liang et al., Molecular Cell, 54:431-444, 2014, featured article on the issue cover). Strikingly, the NTD and dsRBD of Rnt1p function as two rulers for substrate selection. This unusual mechanism represents an example of the evolution of substrate selectivity and provides a framework for understanding the catalytic mechanism of eukaryotic RNase IIIs, including Drosha and Dicer.
