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J. Biol. Chem., Vol. 282, Issue 18, 13824-13832, May 4, 2007

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Structure of Aquifex aeolicus Argonaute Highlights Conformational Flexibility of the PAZ Domain as a Potential Regulator of RNA-induced Silencing Complex Function^*

Umar Jan Rashid, Dirk Paterok, Alexander Koglin, Holger Gohlke^¶, Jacob Piehler, and Julian C.-H. Chen¹

From the Institutes of Biophysical Chemistry, Biochemistry, and ^¶Cell Biology and Neuroscience, J. W. Goethe University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt, Germany

Gene silencing mediated by RNA interference requires the sequence-specific recognition of target mRNA by the endonuclease Argonaute, the primary enzymatic component of the RNA-induced silencing complex. We report the crystal structure of Aquifex aeolicus Argonaute, refined at 3.2Å resolution. Relative to recent Argonaute structures, a 24° reorientation of the PAZ domain in our structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. This rearrangement leads to a branched, Y-shaped system of grooves that extends through the molecule and merges in a central channel containing the catalytic residues. A 5.5-ns molecular dynamics simulation of Argonaute shows a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute monitored by total internal reflection fluorescence spectroscopy shows biphasic kinetics, also indicative of domain rearrangement upon DNA binding. Conformational rearrangement of the PAZ domain may therefore be critical for the catalytic cycle of Argonaute and the RNA-induced silencing complex.

Received for publication, September 6, 2006 , and in revised form, November 21, 2006.

The atomic coordinates and structure factors (code 2NUB) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was funded by the Deutsche Forschungsgemeinschaft SFB 579 and the Hessisches Ministerium für Wissenschaft und Kultur. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and supplemental data.

¹ To whom correspondence should be addressed. Tel.: 49-69-798-29641/30; Fax: 49-69-798-29632; E-mail: chen{at}chemie.uni-frankfurt.de.

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