Poster abstracts
Poster number 21 submitted by Mi Seul Park
Multidomain convergence of Argonaute during RISC assembly correlates with the formation of internal water clusters
Mi Seul Park (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA, Center for RNA Biology ), Raul Araya-Secchi (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA), James A. Brackbill (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA), Hong-Duc Phan (Ohio State Biochemistry Program), Audrey C. Kehling, Ekram W. Abd El-Wahab, Daniel M. Dayeh, Marcos Sotomayor (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA, Center for RNA Biology ), Kotaro Nakanishi (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA, Center for RNA Biology )
Abstract:
In humans, four Argonaute proteins (AGOs) load miRNAs to assemble the RNA-induced silencing complex (RISC) while interacting with TNRC6 proteins that further recruit mRNA degradation complexes for post-transcriptional gene silencing. Previous studies reported that the loading of miRNAs increases the affinity of AGO for TNRC6 proteins, but the molecular mechanism remains elusive. Here, we report the 1.9 Å crystal structure of human Argonaute4 (AGO4) in complex with guide RNA. Our structural comparison with the previously determined apo structure of Neurospora crassa QDE2, an Argonaute homolog, revealed that the PIWI domain is composed of two subdomains, which are fastened with the MID domain upon binding of guide RNA. A mutation at their subdomain interface disrupted TNRC6 binding, suggesting that the conformational change is indispensable for binding to TNRC6 protein. In addition, the current high-resolution structure enabled us to notice that loops and domain linkers wrap 13 and 4 water molecules, forming two clusters inside the AGO4-RISC (We named them LAKEs, Loop-Associated Key Estuaries). Notably, the corresponding water molecules are also found in AGO1 and AGO2 but not in silkworm PIWI protein. Consistent with the observation, the surrounding residues that form hydrogen bonds with the water clusters are conserved throughout eukaryotic AGOs but not PIWIs. The significance of LAKE is evidenced by our in vitro assay that AGO2 lost or lowered slicer activity when their corresponding residue was mutated. Lastly, molecular dynamics simulations show that the water molecules always occupy specific positions at the domain interfaces but are exchangeable with bulk solvent. Altogether, these results suggest that water molecules are essential for maintaining the functional RISC structure after the guide-driven conformational changes, and presumably important also for RISC disassembly when releasing guide RNA.
Keywords: human Argonaute, TNRC6, water molecules