title: Novel Insights into Guide RNA 5'-Nucleoside/Tide Binding by Human Argonaute 2.
abstract: The human Argonaute 2 (hAgo2) protein is a key player of RNA interference (RNAi). Upon complex formation with small non-coding RNAs, the protein initially interacts with the 5'-end of a given guide RNA through multiple interactions within the MID domain. This interaction has been reported to show a strong bias for U and A over C and G at the 5'-position. Performing molecular dynamics simulations of binary hAgo2/OH-guide-RNA complexes, we show that hAgo2 is a highly flexible protein capable of binding to guide strands with all four possible 5'-bases. Especially, in the case of C and G this is associated with rather large individual conformational rearrangements affecting the MID, PAZ and even the N-terminal domains to different degrees. Moreover, a 5'-G induces domain motions in the protein, which trigger a previously unreported interaction between the 5'-base and the L2 linker domain. Combining our in silico analyses with biochemical studies of recombinant hAgo2, we find that, contrary to previous observations, hAgo2 is capable of functionally accommodating guide strands regardless of the 5'-base.
authors.affiliation: Institute of Molecular Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. munishikakalia@gmail.com.|Institute for Neuro- and Bioinformatics, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. munishikakalia@gmail.com.
authors.forename: Munishikha
authors.initials: M
authors.lastname: Kalia
authors.affiliation: Institute of Molecular Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. willkomm@imm.uni-luebeck.de.
authors.forename: Sarah
authors.initials: S
authors.lastname: Willkomm
authors.affiliation: Institute for Neuro- and Bioinformatics, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. j.claussen@jacobs-university.de.
authors.forename: Jens Christian
authors.initials: JC
authors.lastname: Claussen
authors.affiliation: Institute of Molecular Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. restle@imm.uni-luebeck.de.
authors.forename: Tobias
authors.initials: T
authors.lastname: Restle
authors.affiliation: Computational Structural Biology, Bijvoet Center for Biomolecular Research, Faculty of Science-Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. A.m.j.j.bonvin@uu.nl.
authors.forename: Alexandre M J J
authors.initials: AM
authors.lastname: Bonvin
mesh_terms: D000595:Amino Acid Sequence; D060565:Argonaute Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D060565:Argonaute Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C466422:AGO2 protein, human
keywords: MD; RNAi; enzyme kinetics; fluorescence spectroscopy; pre-steady-state kinetics