Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain

1. Structure and nucleic-acid binding of the DrosophilaArgonaute 2 PAZ domain Andreas Lingel, Bernd Simon, Elisa Izaurralde & Michael Sattler European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Nature. 2003 Nov 27;426(6965): 465-9 PDB ID: 1VYN 指導老師:鄒文雄 主講人:蔡榮育 01/21/2005

2. Introduction • RNA interference (RNAi): This evolutionary conserved pathway is triggered in response to exogenous dsRNA introduced to cells. • For example, by viral infection or transfection of in vitro synthesized dsRNA, as well as by the expression of endogenously encoded RNAi triggers, microRNAs (miRNA).

3. RNAi results in the downregulation of a homologous target gene through the cleavage, translational repression, or transcriptional inhibition of its mRNA. • Two core proteins are universally associated with RNAi-related silencing phenomena: Dicer and Argonaute (Ago).

4. Gunter Meister et. al (2004) Nature Vol.431, 343-349 RNA interference pathways in different organismsRISC:RNA-induced silencing complex RITS:RNA-induced transcriptional silencing RNP:ribonucleoprotein rasiRNA:repeat-associated short interfering RNA

5. The siRNA pathway

6. Members of the Dicer and Argonaute protein families are essential components of these RNA-silencing pathways. • These two families possess an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose biochemical function is unknown.

8. View structure

9. Ribbon representation of the Ago2 PAZ domain

11. Biological question • The Argonaute 2 protein (Ago2) is a critical component of RISC. Both Argonaute and Dicer family proteins contain a common PAZ domain whose function is unknown. (1) What is the function of the PAZ domain? (2) Which residue is important?

12. A region comprising the β3, β4, α3 module and the central β-barrel, which together form a clamp-like structure. The presence of aromatic and positively charged residues at this surface may indicate a conserved function in the binding of negatively charged ligands, such as nucleic acids. • The PAZ domain contains a variant of the OB-fold, a module that often binds single-stranded necleic acids ( For example, rho termination factor in transcription ).

13. Surface representation of the PAZ domain coloured by sequence conservation

14. White, blue and red corresponds to neutral, positive and negative electrostatic potential, respectively. Surface representation of the PAZ domain coloured by electrostatic charge

15. Nucleic-acid binding activity of the Ago2 PAZ domain

18. Result • The RNA-binding region involves highly conserved residues, specifically Arg 55 (β 2), Phe 72 (β 3), Thr 80 (β 4), Val 102 (β 5) and Ile 81 (α 3), but also the less conserved Phe 50 in the β 1–β 2 loop. • The results identify Phe 50 and Phe 72 as critical residues for RNA binding. • The PAZ domain also binds single-stranded or double-stranded DNA.

19. 問題探討 • PAZ domain 除了 Phe50 和 Phe72 外,還有哪些 residues 與 RNA 結合?這些 residues 之特性為何? • BLAST 結果呈兩極化,可能之原因為何? • Alignment 結果中其他 conserved residues 可能扮演的角色?

20. Mapping the electrostatic potential on the surface of the protein does not yield an obvious positively charged surface for nucleic acid binding. • The aromatic residues could interact with single-stranded nucleic acids via stacking interactions. • Conserved aromatic residues within the cleft are involved in RNA binding.

21. Stereo view showing one PAZdomain interacting with an siRNA-like end

22. Electrostatic surface of PAZ

23. BLAST

25. Drosophila melanogaster • Anopheles gambiae (瘧蚊) • Homo sapiens • Gallus gallus • Caenorhabditis elegans

26. Alignment Phe72 Arg31 Asn41 Phe50 Phe72 Pro91 Pro47 Pro66