A Single Inter-Domain Salt Bridge within the Human Argonaute 2 Protein Crucially Affects Protein Folding and Consequently Enzymatic Activity

Abstract

RNA interference is a highly complex process involved in posttranscriptional gene regulation. Argonaute (Ago) proteins are the key component of the RNA-induced silencing complex (RISC) and mediate RNA interference (RNAi) in association with small RNAs. Ago2 proteins are composed of four domains: N, PAZ, Mid and PIWI, tethered by L1 and L2 linker regions [1, 2].In the binary complex of Ago2 and single stranded RNA, the 3'-end of the RNA is bound to the PAZ domain and the 5'-phosphate is anchored within a binding pocket in the Mid domain. The PIWI domain harbors the active site, which is composed of a catalytic tetrad as observed in RNase H.In the present study, we performed MD simulations on the hAgo2 protein in complex with a bound miRNA (pdb 4F3T). The protein undergoes prominent breathing motions dominated largely by movements of the PAZ and N domains. Most interestingly, we observed the transient formation a thitherto-undescribed inter-domain salt bridge. The introduction of a mutation preventing formation of this salt bridge drastically affected nucleic acid binding properties of recombinant hAgo2 [3] and abolished the enzymatic activity. In summary, the missing salt bridge not only affects overall protein conformation and stability but also seems to be crucial for proper positioning of the small RNA.1. Elkayam, E., et al., (2012) The structure of human Argonaute-2 in complex with miR-20a. Cell 150(1), 100-110.2. Schirle, N.T. and MacRae, I.J., (2012) The crystal structure of human Argonaute2. Science 336(6084), 1037-1040.3. Deerberg, A., et al., (2013) Minimal mechanistic model of siRNA-dependent target RNA slicing by recombinant human Argonaute 2 protein. PNAS in press.