Structure and nucleic-acid Structure and nucleic-acid binding of the binding of the DrosophilaDrosophilaArgonaute 2 PAZ domainArgonaute 2 PAZ domain

Andreas Lingel, Bernd Simon, Elisa Izaurralde & Andreas Lingel, Bernd Simon, Elisa Izaurralde & Michael SattlerMichael Sattler

European Molecular Biology Laboratory, Meyerhofstrasse European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany1, D-69117 Heidelberg, Germany

Nature. 2003 Nov 27;426(6965): 465-9Nature. 2003 Nov 27;426(6965): 465-9

PDB ID: 1VYNPDB ID: 1VYN

指導老師指導老師 :: 鄒文雄鄒文雄主講人主講人 :: 蔡榮育蔡榮育

01/21/200501/21/2005

IntroductionIntroduction

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).

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).

RNA interference pathways in different organismsRNA interference pathways in different organisms

RISC:RNA-induced silencing complex RITS:RNA-induced transcriptional RISC:RNA-induced silencing complex RITS:RNA-induced transcriptional silencing silencing

RNP:ribonucleoprotein rasiRNA:repeat-associated short interfering RNP:ribonucleoprotein rasiRNA:repeat-associated short interfering RNARNA

Gunter Meister et. al (2004) Nature Vol.431, 343-349Gunter Meister et. al (2004) Nature Vol.431, 343-349

The siRNA pathwayThe siRNA pathway

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.

View structureView structure

Ribbon representation Ribbon representation of the Ago2 PAZ domainof the Ago2 PAZ domain

Biological questionBiological 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?

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 ).

Surface representation of the PAZ domain coloured by sequence conservation

Surface representation of the PAZ domain coloured by

electrostatic charge

White, blue and red corresponds to neutral, positive and negative electrostatic potential, respectively.

Nucleic-acid binding activity of the Ago2 PAZ domain

ResultResult The RNA-binding region involves highly The RNA-binding region involves highly

conserved residues, specifically Arg 55 (β conserved residues, specifically Arg 55 (β 2), Phe 72 (β 3), Thr 80 (β 4), Val 102 (β 2), Phe 72 (β 3), Thr 80 (β 4), Val 102 (β 5) and Ile 81 (α 3), but also the less 5) and Ile 81 (α 3), but also the less conserved Phe 50 in the β 1–β 2 loop.conserved Phe 50 in the β 1–β 2 loop.

The results identify Phe 50 and Phe 72 as The results identify Phe 50 and Phe 72 as critical residues for RNA binding.critical residues for RNA binding.

The PAZ domain also binds single-The PAZ domain also binds single-stranded or double-stranded DNA.stranded or double-stranded DNA.

問題探討問題探討 PAZ domain PAZ domain 除了 除了 Phe50 Phe50 和 和 Phe72 Phe72 外外 ,,

還有哪些 還有哪些 residues residues 與 與 RNA RNA 結合結合 ?? 這些 這些 residues residues 之特性為何之特性為何 ??

BLAST BLAST 結果呈兩極化結果呈兩極化 ,, 可能之原因為何可能之原因為何 ??

Alignment Alignment 結果中其他 結果中其他 conserved conserved residues residues 可能扮演的角色可能扮演的角色 ??

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.

Stereo view showing one PAZdomain interacting with an

siRNA-like end

Electrostatic surface of PAZ

BLASTBLAST

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

AlignmentAlignment

Phe50

Phe72

Phe72Asn41

Pro91

Arg31

Pro47 Pro66