| The piRNA pathway is responsible for silencing of transposons and protects the genome from disruption by these mobile elements during germ line development. Piwi proteins interact with piRNAs, which differ from the other commonly found small RNAs, siRNAs and miRNAs, in length, averaging 26-31 nt, and are thought to originate from long primary transcripts clustered throughout the genome. In addition, piRNAs are methylated on the 2' oxygen of the 3' terminal ribose by a methyltransferase. The PAZ domain is a conserved domain found in the Argonaute family of proteins that is responsible for anchoring the 3' end of the guide strand. We determined the crystal structure of the PAZ domain from the human protein Hiwi, a member of the Piwi subfamily, to a resolution of 1.50 A. The structure reveals that Hiwi PAZ shares the conserved pocket for 3' terminal residues of RNA found in other PAZ domains. Hiwi PAZ binding to RNA was characterized by gel filtration chromatography, NMR and surface plasmon resonance. Binding affinity data showed that Hiwi PAZ binds RNA in the micromolar range, preferring single-stranded RNA over double-stranded RNA, and, notably, prefers RNA that is 3' terminal 2'-O-methylated. The structure of the 3' binding pocket can accommodate a 3' terminal 2'-O-methyl modification, which was visualized in a docked model of the complex. In comparison with the homologous pocket in the hAgo1 PAZ:RNA structure, the Hiwi PAZ pocket is larger and has hydrophobic side chains positioned to run alongside the 2'-O-methyl group. In addition, the Piwi subfamily PAZ domains contain a variable loop, between beta6--beta7, of length 12-15 amino acid residues, compared to approximately 5 residues in the Argonaute subfamily and 35 residues in Dicer. This loop is P/G and K/R rich, suggesting that it may be both highly flexible and involved in phosphate backbone interactions. While structural information about the loop was not available in the crystal structure, backbone dynamics studies via NMR revealed that the loop remains flexible upon PAZ binding to RNA, suggesting possible roles in binding preference for flexible ssRNA or other protein-protein interactions. Finally, computational docking---with loop and RNA flexibility allowed in both partners---was used to evaluate possible conformations of the Hiwi PAZ:RNA complex and was compared with the homologous hAgo:RNA complex structure. Together with binding data and loop backbone flexibility, these results suggest that maintaining loop flexibility serves to aid Hiwi PAZ in binding preferentially to ssRNA. |
