The Making of a Slicer: Activation of Human Argonaute-1

The Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 miRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved DEDH catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. hAgo3, with an intact tetrad, could be activated by swapping in the N domain of hAgo2, without additional PIWI domain mutations. An additional mutation on a loop adjacent to the active site of hAgo1 results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme. RNAsprofiling of small RNAs associated with recombinant protein used for chrystallographic studies