Article

Crystal Structure of Silkworm PIWI-Clade Argonaute
Siwi Bound to piRNA
Graphical Abstract

Authors
Naoki Matsumoto, Hiroshi Nishimasu,
Kazuhiro Sakakibara, ..., Haruhiko Siomi,
Mikiko C. Siomi, Osamu Nureki

Correspondence
nisimasu@bs.s.u-tokyo.ac.jp (H.N.),
siomim@bs.s.u-tokyo.ac.jp (M.C.S.),
nureki@bs.s.u-tokyo.ac.jp (O.N.)

In Brief
The first structure of the PIWI protein
reveals mechanistic insights into its
target recognition and cleavage.

Highlightsd

Crystal structure of a PIWI-clade Argonaute bound to piRNAd

Structural basis for the preference of Siwi for the guide 1UddSpecific recognition mechanism of the 50 and 30 ends of thebound piRNA by Siwi
Structural similarities and differences between AGO- andPIWI-clade Argonautes

Matsumoto et al., 2016, Cell 167, 484–497
October 6, 2016 ª 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.09.002

Data Resources
5GUH

Article
Crystal Structure of Silkworm PIWI-CladeArgonaute Siwi Bound to piRNA
Naoki Matsumoto,1,4 Hiroshi Nishimasu,1,2,4,* Kazuhiro Sakakibara,1 Kazumichi M. Nishida,1 Takamasa Hirano,3
Ryuichiro Ishitani,1 Haruhiko Siomi,3 Mikiko C. Siomi,1,* and Osamu Nureki1,5,*
1Graduate School of Science, Department of Biological Sciences, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032,
Japan
2JST, PRESTO, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
3Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
4Co-first author
5Lead Contact*Correspondence: nisimasu@bs.s.u-tokyo.ac.jp (H.N.), siomim@bs.s.u-tokyo.ac.jp (M.C.S.), nureki@bs.s.u-tokyo.ac.jp (O.N.)
http://dx.doi.org/10.1016/j.cell.2016.09.002

SUMMARYPIWI-clade Argonaute proteins associate with PIWIinteracting RNAs (piRNAs) and silence transposable
elements in animal gonads. Here, we report the crystal structure of a silkworm PIWI-clade Argonaute,Siwi, bound to the endogenous piRNA, at 2.4 Å resolution. Siwi adopts a bilobed architecture consistingof N-PAZ and MID-PIWI lobes, in which the 50 and
30 ends of the bound piRNA are anchored by the
MID-PIWI and PAZ domains, respectively. A structural comparison of Siwi with AGO-clade Argonautes
reveals notable differences in their nucleic-acidbinding channels, likely reflecting the distinct lengthsof their guide RNAs and their mechanistic differencesin guide RNA loading and cleavage product release.
In addition, the structure reveals that Siwi and prokaryotic, but not eukaryotic, AGO-clade Argonautes
share unexpected similarities, such as metal-dependent 50 -phosphate recognition and a potential structural transition during the catalytic-tetrad formation.
Overall, this study provides a critical starting point
toward a mechanistic understanding of piRNA-mediated transposon silencing.
INTRODUCTION
Argonaute proteins associate with small RNA guides and form
RNA-induced silencing complexes (RISCs), which repress target
RNAs complementary to the guide RNAs at the transcriptional or
posttranscriptional level (Siomi and Siomi, 2009; Ghildiyal andZamore, 2009; Czech and Hannon, 2011; Meister, 2013). The
Argonaute family proteins can be classified into two clades,
AGO and PIWI. The AGO-clade proteins are ubiquitously expressed in most organisms and associate with 22 nucleotide(nt) microRNAs (miRNAs) or small interfering RNAs (siRNAs) toform RISCs (Siomi and Siomi, 2009; Ghildiyal and Zamore,
2009; Czech and Hannon, 2011; Meister, 2013). The RISCs
repress the expression of their target genes and regulate a
484 Cell 167, 484–497, October 6, 2016 ª 2016 Elsevier Inc.

wide variety of biological processes (Okamura and Lai, 2008).
In contrast, the PIWI-clade proteins are primarily expressed inanimal gonads and associate with 23–33 nt PIWI-interacting
RNAs (piRNAs) to form piRNA-induced silencing complexes(piRISCs) (Malone and Hannon, 2009; Weick and Miska, 2014;
Iwasaki et al., 2015; Czech and Hannon, 2016). The piRISCs
repress the expression of transposable elements and thus
contribute to the maintenance of genome integrity. There arenotable differences between the RISC and piRISC assembly
processes. During the RISC assembly, a small RNA duplex isloaded onto the AGO protein (Kobayashi and Tomari, 2016).
Subsequently, one strand of the duplex (the passenger strand)
is selectively discarded, and the other strand (the guide strand)
remains bound to the AGO protein to form the mature RISC. In
contrast, during the piRISC assembly, a long single-stranded
piRNA intermediate is initially loaded onto the PIWI protein,with its 50 end anchored within the protein. Subsequently, thepiRNA intermediate in the PIWI protein is cleaved on the 30 downstream side by the endonuclease Zucchini (MitoPLD in mouse) toform a precursor piRNA (pre-piRNA) (Ipsaro et al., 2012; Nishimasu et al., 2012; Han et al., 2015; Mohn et al., 2015; Homolka
et al., 2015). Any extra 30 nucleotides of the pre-piRNA boundto the PIWI protein are trimmed by the exonucleases Nibbler(Feltzin et al., 2015; Wang et al., 2016) and/or Trimmer (Izumi
et al., 2016; Tang et al., 2016). The piRNA 30 end is then 20 -Omethylated by Hen1 (also known as Pimet) to increase its stability (Horwich et al., 2007; Saito et al., 2007).
Previous structural studies of the prokaryotic and eukaryotic
AGO-clade proteins provided mechanistic insights into RISCmediated RNA silencing. The crystal structures of Pyrococcus
furiosus AGO (Song et al., 2004) and Aquifex aeolicus AGO(Yuan et al., 2005) in their apo states revealed that Argonaute
adopts a bilobed architecture, comprising four signature domains (N, PAZ, MID, and PIWI) and two linker domains (L1 andL2), and that the PIWI domain adopts an RNase H fold and is
responsible for target cleavage. Subsequently, the crystal structures of Thermus thermophilus AGO (TtAgo) bound to a guidestrand (Wang et al., 2008a) and bound to guide and target strands(Wang et al., 2008b, 2009; Sheng et al., 2014) provided mechanistic insights into the guide-target recognition and the subsequent target cleavage. The TtAgo structures showed that the50 and 30 ends of the guide are anchored within the MID-PIWI andPAZ domains, respectively. More recently, the crystal structuresof eukaryotic AGOs bound to a guide RNA, such as Kluyveromyces polysporus Argonaute (KpAgo) (Nakanishi et al., 2012), human Argonaute2 (hAgo2) (Schirle and MacRae, 2012; Elkayam
et al., 2012), and human Argonaute1 (hAgo1) (Faehnle et al.,2013; Nakanishi et al., 2013), have been reported. These structures highlighted the structural differences between the eukaryotic and prokaryotic AGOs, such as the eukaryotic-specificinsertions, despite their overall structural similarity. Furthermore,
a series of crystal structures of hAgo2 bound to a guide RNA anda target RNA revealed the conformational changes in the protein
accompanying the guide-target base pairing (Schirle et al., 2014).
Single-molecule imaging studies further illuminated the structural dynamics of target recognition and cleavage by mammalian
Ago2 (Chandradoss et al., 2015; Jo et al., 2015; Salomon et al.,2015) and Drosophila Ago2 (Yao et al., 2015). In stark contrast
to the AGO-clade Argonautes, structural information is still limited
for the PIWI-clade Argonautes. Although the structures of the
PAZ domain (Zeng et al., 2011; Tian et al., 2011; Simon et al.,2011) and the MID domain (Cora et al., 2014) of the mammalian
PIWI proteins are available, the structures of full-length
PIWI-clade Argonautes remain unresolved, primarily because ofdifficulties in sample preparation for structural analysis.
BmN4 cells, a Bombyx mori ovary-derived cultured cell line,
express two PIWI proteins, Siwi and BmAgo3 (Kawaoka et al.,2009), and are widely used as a cultured cell model for investigating the mechanisms underlying the piRNA pathway. In thesecondary piRNA biogenesis pathway (also known as the pingpong cycle) (Brennecke et al., 2007; Gunawardane et al., 2007;
Czech and Hannon, 2016), antisense piRNA-bound Siwi andsense piRNA-bound BmAgo3 reciprocally cleave complementary transcripts, to couple the amplification of piRNAs with therepression of transposable elements (Kawaoka et al., 2009;
Xiol et al., 2014; Nishida et al., 2015). In this process, Siwi requires the ATP-dependent RNA unwinding activity of the
DEAD-box RNA helicase BmVasa, for the release of the cleavage
products (50 and 30 cleavage products) (Nishida et al., 2015). Thismechanism ensures the transfer of the 30 cleavage products
from Siwi to BmAgo3, thereby facilitating the ping-pong cycle(Xiol et al., 2014; Nishida et al., 2015).
In this study, we purified the endogenous Siwi-piRISC from
BmN4 cells, using a monoclonal anti-Siwi antibody (Nishida
et al., 2015), and determined its crystal structure at 2.4 Å resolution. The structure reveals that Siwi adopts a bilobed architecture consisting of the N-PAZ and MID-PIWI lobes, with the 50
and 30 ends of the co-purified endogenous piRNA anchored at
the MID-PIWI interface and the PAZ domain, respectively. Our
structure-guided mutational analyses confirm that the anchoringof both ends of the piRNA is important for stable piRISC formation and reveal that the catalytic tetrad in the PIWI domain is
responsible for target cleavage. A structural comparison ofSiwi and hAgo2 revealed notable differences in the arrangementof the N-PAZ lobes with respect to the MID-PIWI lobes, resulting
in the structural differences in their central channels that accommodate the guide and target RNAs. Collectively, the present
structure provides critical information toward a mechanistic understanding of piRISC-mediated transposon silencing.

RESULTS
Preparation of the Siwi-piRISC
We prepared Sepharose beads conjugated with the anti-Siwi
antibody (Nishida et al., 2015) and set out to purify the endogenous Siwi-piRISC from BmN4 cells for crystallization. Importantly, previous studies showed that the hAgo2-RISC, but not
the small RNA-free hAgo2, is resistant to protease (thermolysin)
digestion (Elkayam et al., 2012) and that the anti-Siwi antibody
recognizes the N-terminal intrinsically disordered region of Siwi
(Nishida et al., 2015). Based on these observations, we hypothesized that the Siwi-piRISC core is also resistant to proteasetreatment and could be separated from the anti-Siwi antibody
beads by the protease digestion of the N-terminal disordered region of Siwi (Figure S1A). To test this hypothesis, we immunoisolated the Siwi-piRISC from BmN4 lysates using the anti-Siwi
antibody beads, treated the complex with thermolysin, andthen analyzed the supernatant by SDS-PAGE. Whereas theimmunoisolated full-length Siwi (101 kDa) migrated as an
100-kDa band on the gel, an 80-kDa single band was liberated in the supernatant after the thermolysin treatment (Figure S1B). An N-terminal sequence analysis revealed that the80-kDa fragment begins with Ile130 of Siwi, suggesting that itcorresponds to the stable core region (residues 130–899) of
Siwi. The Siwi core region was further purified by chromatography on heparin and size-exclusion columns and was eluted
as a single peak from the size-exclusion column (Figures S1C
and S1D). Absorbances at 260 and 280 nm of the peak indicatedthat the purified Siwi associates with nucleic acids (Figure S1C).
32
P-end labeling of the RNAs isolated from the fraction revealed
that 28- to 30-nt RNAs are associated with Siwi (Figure S1E),
consistent with our previous observation (Nishida et al., 2015).
Together, these results indicate that the purified sample represents the mature Siwi-piRISC core, which lacks its N-terminal
flexible region.
Overall Structure of the Siwi-piRISC
We crystallized the purified Siwi-piRISC and determined its
structure at 2.4 Å resolution, by molecular replacement using
the hAgo2 structure (PDB: 4OLA) (Schirle and MacRae, 2012)
as the search model (Table S1). The structure revealed that
Siwi (residues 130–899) consists of four domains (N, PAZ, MID,
and PIWI) and three linker domains (L0, L1, and L2) (residues
130–167 are referred to as L0) and can be divided into two lobes(N-PAZ and MID-PIWI) (Figures 1A and 1B). The N-PAZ lobe consists of the L0 (residues 157–167), N (residues 168–240), L1(residues 241–317), PAZ (residues 318–426), and L2 (residues
427–484) domains, whereas the MID-PIWI lobe consists of theL0 (residues 130–156), L2 (residues 485–527), MID (residues
528–657), and PIWI (residues 658–899) domains. The L0 region
contains two b strands and interacts with the b strands in the
L1, L2, and PIWI domains, thereby contributing to the integrationof the two lobes (Figure 1B). Siwi has a nucleic-acid-binding
channel between the two lobes, in which electron densities areobserved for the co-purified endogenous RNAs (Figure S2A),
as in the crystal structures of the eukaryotic AGOs (Schirle and
MacRae, 2012; Nakanishi et al., 2012; Faehnle et al., 2013; Nakanishi et al., 2013). The electron density for the 50 nucleotide
Cell 167, 484–497, October 6, 2016 485

MID-PIWI lobe

A
1

130

ID

168

L0

241

N

318

427

L1

528

PAZ

L2

658

899

MID

PIWI

N-PAZ lobe
Crystallized core region

Siwi

B
MID

PAZ

90°
C

L2
5′
N

3′

piRNA

N

PIWI

90°

L1

N

L0
N-PAZ lobe

MID-PIWI lobe

C

hAgo2

PAZ

Siwi / hAgo2

D
MID

MID

PAZ

3′

L2

L2
miRNA

5′

N
N

PIWI

PIWI
L0

L0

L1
MID-PIWI lobe

L1

N-PAZ lobe

Figure 1. Overall Structure(A) Domain organization of Siwi. ID, intrinsically disordered region.
(B) Crystal structure of the Siwi-piRISC. Top and bottom views are shown on the right.
(C) Crystal structure of the hAgo2-RISC (PDB: 4W5N) (Schirle et al., 2014).
(D) Superimposition of Siwi (colored) and hAgo2 (PDB: 4W5N) (beige). Structural differences in their N and PAZ domains are indicated by red arrows.
See also Figures S1 and S2 and Table S1.

486 Cell 167, 484–497, October 6, 2016

could be fitted to a uridine (Figure S2B), consistent with the preference of Siwi for the U1 nucleotide in the guide piRNA (g1U bias)(Kawaoka et al., 2009, 2011; Xiol et al., 2014; Nishida et al.,2015). The electron density was observed for the 20 -O-methylgroup in the 30 nucleotide (Figure S2C), consistent with earlier
observations that mature piRNAs possess the 20 -O-methylated
30 end (Horwich et al., 2007; Saito et al., 2007; Kawaoka et al.,2011). Since the Siwi-bound endogenous piRNAs have divergent
sequences and lengths (predominantly 28-nt long) (Kawaoka
et al., 2009; Nishida et al., 2015), we modeled 1-UAUUU-5 and
26-UUUm-28 (Um indicates the 20 -O-methylated U) as the 50
and 30 segments of the bound piRNA, respectively, according
to the size and shape of their nucleobases. No electron density
was observed for the middle segment (nucleotides 6–25) of thepiRNA, as in the guide-bound eukaryotic AGO structures (Schirleand MacRae, 2012; Nakanishi et al., 2012, 2013; Elkayam et al.,2012; Faehnle et al., 2013), suggesting that the middle region isdisordered in the present structure. Together, these observations indicate that the present structure represents the SiwipiRISC bound to mature piRNAs.

Met347/Thr354 and Leu313 residues of Siwi are replaced with
Glu268/Lys278 and Phe224 in hAgo2, respectively, and the
AGOs have a conserved proline residue (Pro229 in hAgo2) inthe L1 domain (Figure S5). These residues of hAgo2 appear toprevent the L1 and PAZ domains from forming a b barrel-likestructure (Figures 2D and 2F). Instead, hAgo2 has Tyr225 in
the last b strand (b12) of the L1 domain, which forms a hydrophobic core with residues from the PAZ domain (Leu265 andPhe233) and the L2 domain (Gln350 and Cys352) (Figure 2F).
In addition, the L1-PAZ-L2 interface is reinforced by polar interactions between Arg196 (L1) and Glu261 (PAZ), and between
Gln350 (L2) and Lys266 (PAZ) (Figure 2F). The Phe224/Pro229/
Gln350 and Arg196/Tyr225/Glu261 residues of hAgo2 are strictly
and highly conserved among the AGOs, but not among the
PIWIs, respectively (Figure S5). Indeed, these interactions
at the L1-PAZ-L2 interface are conserved in hAgo1 (Faehnle
et al., 2013; Nakanishi et al., 2013). Together, these structuralobservations highlight the distinct spatial arrangements of the
N and PAZ domains relative to the L1-L2 domains between the
AGO- and PIWI-clade Argonautes.

Structural Comparison between Siwi and hAgo2
A structural comparison between Siwi and hAgo2 (Schirle and
MacRae, 2012; Elkayam et al., 2012; Schirle et al., 2014) revealed unexpected similarities and differences between the
PIWI and AGO proteins (Figures 1B–1D). The individual domainsof Siwi and hAgo2 can be superimposed (root-mean-square deviation of 0.9–2.0 Å for equivalent Ca atoms) (Figure S3A),
although they share limited sequence identities (13%–32%) (Figure S4). However, there are notable differences in the arrangement of their N and PAZ domains relative to the rest of the protein(Figure 1D). In addition, the relative arrangement between their N
and PAZ domains differs between Siwi and hAgo2 (Figure S3B).
These distinctive arrangements are stabilized by extensive interactions at their N-L1-L2 and L1-PAZ-L2 interfaces (Figure 2).
At the N-L1-L2 interface of Siwi, Trp166 in the L0 domain and
Trp477 in the L2 domain form a hydrophobic core with residues
from the N domain (Leu168, Leu200, Tyr207, and Val209) and theL1 domain (Tyr247, Phe251, and Ile254), thereby playing a central role in the formation of the N-L1-L2 interface (Figures 2A
and 2B). Notably, Trp477 of Siwi is conserved among the PIWIs,
but not among the AGOs, in which the equivalent residue isconserved as phenylalanine (Phe397 in hAgo2) (Figure S5). In
the hAgo2 structure (Schirle et al., 2014), Phe397 forms a hydrophobic core with residues from the N domain (Ile54, Val93,
Tyr101, and Ala103) and the L1 domain (Val165), in a manner
distinct from that of Siwi Trp477 (Figures 2D and 2E). In addition,hAgo2 has an AGO-specific a-helix (referred to as AGO-Ins) between the N and L1 domains that contributes to the N-L1 interface formation (Figures 2D, 2E, and S5).
In the L1-PAZ-L2 interface of Siwi, four b strands (b8, b9, b11,
and b12) in the L1 domain and three b strands (b13, b14, and b19)in the PAZ domain form a seven-stranded b-barrel-like structure,
which is stabilized by a hydrophobic core formed by residues
from the L1 domain (Met267, Phe272, Pro274, Trp288, Lys311,
and Leu313) and the PAZ domain (Met347, Thr354, andThr426) (Figures 2A and 2C). This b-barrel-like structure fixes
the arrangement between the L1 and PAZ domains. The

Recognition of the 50 Phosphate of the piRNA
The 50 end of the piRNA is flipped into a binding pocket atthe interface between the MID and PIWI domains (Figure 3A).
The 50 phosphate is recognized by the main-chain amide groupof Val624 and the side chains of Tyr607, Lys611, Gln623, andLys649 (Figure 3A). We observed a strong electron density inthe vicinity of the 50 phosphate; we modeled it as a Mg2+ ion,based on its octahedral geometry and coordination distances(2.1 Å) (Figure S6A). The Mg2+ ion was likely co-purified fromthe BmN4 cells, since the purification and crystallization buffers
lacked divalent cations. The Mg2+ ion is octahedrally coordinated by phosphates 1 and 3 in the piRNA, the side chain of
Gln645, the C-terminal carboxylate group of Leu899, and a watermolecule (Figure 3A). To examine the functional importanceof the residues involved in the 50 phosphate recognition, we
expressed the FLAG-tagged, wild-type and mutants of Siwi
in BmN4 cells, immunopurified the Siwi proteins, and thenanalyzed the Siwi-bound piRNAs (Figure 3B). The K611A,
Q623A, Q645A, and K649A mutants were associated with
substantially reduced levels of piRNAs, as compared withwild-type Siwi. These results confirmed the importance of the
50 phosphate recognition for piRNA loading, consistent withprevious studies (Kawaoka et al., 2011; Cora et al., 2014).
Tyr607, Lys611, Gln623, Gln645, and Lys649 are strictlyconserved among the PIWI-clade proteins (Figure S5), indicating
the conservation of the metal-dependent recognition mechanism of the piRNA 50 phosphate among them. Notably, Gln645
of Siwi is replaced with a lysine in the eukaryotic AGOs
(Lys566 of hAgo2) (Figure S5), and Lys566 interacts with phosphates 1 and 3 of the guide RNA in the hAgo2 structure (Schirleand MacRae, 2012; Elkayam et al., 2012) (Figures 3C and 3D). In
contrast, in the TtAgo structure, phosphates 1 and 3 in the guideare recognized by the protein in a metal-dependent manner with
the Mg2+ ion held by Gln433, which is equivalent to Gln623 ofSiwi (Gln645 of Siwi is replaced with Gly453 in TtAgo) (Shenget al., 2014) (Figure 3E). These structural findings indicate that
the PIWIs and prokaryotic AGOs recognize the 50 phosphate ofCell 167, 484–497, October 6, 2016 487

A

Siwi

B

L2

L2

2
I253

L1

254
4
I254

2
I253

L1

L200

I254
254
4

W47
77
7
7
W477

PAZ

I250
250

2
I250
F251
1

L2

F251

Y207
7
Y207

Y247

L1

Y207
Y207
7

Y247
Y

L1
L
1
L168

L168
1
V209
V
V2

V
V2
V209

L0

L200
L

77
W477

L0

L0
V240
P242

V240
V24
V240
P242

W1
W166

N

W166
W1

N

N

150°

C
PAZ

PAZ
L2

PAZ

M347
47
7

L2

M347
47
7

T42
T426
L313
L3
L
3
313

T
354
4
T354

L1

M267

N

L1

D

hAgo2

L313
L313
L31
L3

M267
7

288
88
W288

K311
K 11
K31
W288
W288

F272

L1

P274
4

E

T42
T426

T354
4
T354

K311
K
311
1

F272

L0

L2

P274
4

L2

L2

V164
V
1
164
4

V164
4
V164
V
V93

PAZ
L1

V
V93

L1

Y101

V165

I159
1

L0

L162
L
1
A161

L2
N

M213

L140

L1
L
162
L162
A161

F397
A103
A10
4
I54
M213
L147

T158
T1
8

N

L143

7
F397
A103
A10
54
I54

V138

L0
L147

Y101
Y101

I159
1

L0

L1

V165

L147

L140

V138
V1
V138

T158

N

L143

AGO-Ins

AGO-Ins

AGO-Ins 150°

F

E261
2 1

PAZ

L265
L2

E261
261

PAZ

F233
P22
P229

L265
L2

L2
Y22
Y225

K266
K2

I346
34

L2
Y22
Y225

R196

K266

C352

Q350

PAZ

F233
P22
P229

34
I346

R196
C352

Q350
Q35

L2

N

L1

L0
AGO-Ins

L1

F224

F224

W1
W199

W1
W199

F182
4
A184

L1

F182
A184

Figure 2. N-PAZ Lobe
(A) N-PAZ lobe of Siwi.
(B and C) N-L1-L2 (B) and L1-PAZ-L2 (C) interfaces of Siwi (stereoviews).
(D) N-PAZ lobe of hAgo2 (PDB: 4W5N) (Schirle et al., 2014).
(E and F) N-L1-L2 (E) and L1-PAZ-L2 (F) interfaces of hAgo2 (PDB: 4W5N) (stereoviews). The AGO-specific insertion is highlighted in yellow.
See also Figures S3, S4, and S5.

the guide strand in metal-dependent manners, whereas the eukaryotic AGOs neutralize the negative charge of the 50 phosphate
via a conserved lysine residue.
488 Cell 167, 484–497, October 6, 2016

Recognition of g1U of the piRNA
The U1 nucleotide in the piRNA (g1U) is recognized in a basespecific manner by a loop region between the third b strand

R606

D605

Y603

D605

R606

Y603

N602
Y607

N602

60 -

Y607

C626

C626

N629

U1
K611

(nt)
100 90 80 70 -

MID

K611

50 -

N629

U1

V624

40 -

V624
Q623

Q623
2

3

2

3

K649

30 -

- piRNA

K649

Q645
L899 (C)PIWI- FLAG-Siwi

Q645
L899 (C)

PIWI

C

D

hAgo2 (g1U)
P527

K649A

P576

MID

WT

P576

D574

Q645A

Siwi

D574

Q623A

B

Siwi

K611A

A

E

hAgo2 (g1A)

T526

P523

P527

MID

TtAgo
P523

T526

MID

MID
M417

Q548

Q548

Y529

N436

Y529
A1

U1

T1

N551
C546

K533

2

Q545

R418

2

K422

I434

Q545

R812

3

3

K566

Q433

K566
R792

R792

PIWI

A859 (C)

F

2
3

K457

K570

K570

PIWI

N551
C546

K533

G453
V685 (C)PIWI

A859 (C)

G
Siwi

hAgo2

L2

α7

L2

α7
1

1
F436

I365

5

3
4

7

I638

3

6

5

4
2

2

T559

Y804
R761
N859
R792

Q871
Y857

PIWI

I756
S798

MID
H753

PIWI

MID

Y790

Figure 3. Recognition of the piRNA 50 Segment(A) Recognition of the piRNA 50 segment by Siwi (stereoview). The bound Mg2+ ion and water molecule are shown as magenta and red spheres, respectively.
Hydrogen bonding and electrostatic interactions are indicated by cyan dashed lines. Coordinations to the Mg2+ ion are shown as gray dashed lines.
(B) piRNA loading in the wild-type and mutants of Siwi. FLAG-Siwi was used as a loading control.
(C–E) Recognition of the guide 50 segment by hAgo2 (g1U; PDB: 4W5O) (C), hAgo2 (g1A; PDB: 4OLA) (D), and TtAgo (g1T; PDB: 4NCB) (E). In (E), the bound Mg2+
ion is shown as a magenta sphere.
(F and G) Recognition of the seed region in the guide RNA by Siwi (F) and hAgo2 (PDB: 4W5N) (G).
See also Figure S6 and Table S2.

Cell 167, 484–497, October 6, 2016 489

and the third a-helix in the MID domain (known as the specificity loop [Frank et al., 2010]) (Figure 3A). The N3 of the U1forms a hydrogen bond with the main-chain carbonyl groupof Tyr603 in the specificity loop, and its nucleobase stacks
with the side chain of Tyr607 (Figure 3A). The conformation of
the specificity loop appears to be stabilized by stacking interactions between Pro576-Arg606-Tyr603, with Arg606 forming
salt bridges with Asp574 and Asp605 (Figure 3A). Modelingindicated a steric clash or charge repulsion between Tyr603
in the specificity loop and the modeled A, G and C (Figure S6B),
thus explaining the g1U bias of Siwi (Kawaoka et al., 2009,2011; Xiol et al., 2014; Nishida et al., 2015). The residues inthe specificity loop differ between Siwi and BmAgo3 (Figure S5),
consistent with the observation that, unlike Siwi, BmAgo3 lacks
the preference for the piRNA 50 nucleotide (Kawaoka et al.,2009, 2011; Nishida et al., 2015). Although Drosophila Piwi
and Aubergine also prefer g1U (Brennecke et al., 2007), theresidues in their specificity loops differ from those in Siwi (Figure S5). Thus, further structural studies are required to elucidate their g1U recognition mechanisms. hAgo2 preferentiallybinds g1U and g1A (Frank et al., 2010; Elkayam et al., 2012),
and g1U or g1A is accommodated within a pocket defined by
the specificity loop in the hAgo2 structure (Frank et al., 2010;
Schirle and MacRae, 2012; Elkayam et al., 2012; Schirle
et al., 2014) (Figures 3C and 3D). The specificity loop ofhAgo2 contains Pro523, Gly524, and Pro527, which are highly
conserved among the eukaryotic AGOs, but not among the
PIWIs (Figure S5); it adopts a conformation distinct from thatof Siwi (Figures 3C and 3D). Together, these structural observations reinforce the notion that the preference for the guide 50
nucleotide is primarily defined by the specificity loop in the
MID domain.
Recognition of the Seed Region of the piRNAIn the Siwi structure, nucleotides 2–5 of the piRNA adopt a nearly
A-form conformation (Figure S6C), and are bound to a narrowgroove formed by the L2, MID and PIWI domains (Figures 3F
and S6D), as in the eukaryotic AGOs (Schirle and MacRae,
2012; Nakanishi et al., 2012, 2013; Elkayam et al., 2012; Faehnle
et al., 2013). This structural feature indicated that, as in theAGOs, nucleotides 2–5 (the seed region) in the piRNA serve asa nucleation site for base pairing with a target RNA. Base 2 interacts with Ile638 in the MID domain, whereas phosphates 4 and 5
are recognized by Tyr857 and Gln871 in the PIWI domain,
respectively (Figure 3F). The 20 -OH group of nucleotide 3 interacts with the main-chain carbonyl group of Asn859. In addition,base 5 interacts with Phe436 in helix a7 in the L2 domain (Figure 3F), indicating that the base stacking between nucleotides
5 and 6 would be interrupted. In the guide-bound hAgo2 structure (Schirle and MacRae, 2012; Elkayam et al., 2012; Schirle
et al., 2014), Ile365 in helix a7, which is equivalent to helix a7 inSiwi, is inserted between nucleotides 6 and 7 of the guideRNA, thereby inducing a pronounced kink in the guide strand(Figure 3G). Moreover, a comparison between the guide-boundand guide-target-bound hAgo2 structures revealed that helix
a7 in the L2 domain is displaced outward upon guide-target
base pairing, thus widening the nucleic-acid-binding channel(Schirle et al., 2014). Phe436 of Siwi is conserved among the
490 Cell 167, 484–497, October 6, 2016

PIWIs, but not among the AGOs, whereas Ile365 of hAgo2 isconserved among the AGOs, but not among the PIWIs (Figure S5). These observations imply that Phe436 of Siwi andIle365 of hAgo2 play similar roles in inducing a kink in the guideRNA within the binary complex. Together, these structural findings suggest that, as in mammalian Ago2 (Chandradoss et al.,2015; Jo et al., 2015; Salomon et al., 2015), Siwi primarily usesa small region (nucleotides 2–5) of the piRNA guide to interrogate
their target sites, and the propagation of the guide-target pairing
induces the widening of the nucleic-acid-binding channel.
Recognition of the 30 End of the piRNA
The 30 end of the piRNA is anchored within a binding pocket in
the PAZ domain (Figure 4A). The 20 -OH and phosphate groupsof nucleotide 27 interact with Tyr355 and Lys386, respectively(Figure 4B). Base 28 forms a stacking interaction with Phe370,
and phosphate 28 is extensively recognized by Tyr350, Tyr355,
Tyr382 and Tyr387 (Figure 4B). The 30 -OH group and the 20 -oxygen atom of nucleotide 28 form hydrogen bonds with Tyr383/
Tyr417 and Tyr417, respectively (Figure 4B). Importantly, the20 -O-methyl group of nucleotide 28 is accommodated within
a hydrophobic pocket formed by Phe370, Tyr379 and Tyr417(Figure 4C), as in the isolated PAZ domains from Hiwi1 (Tianet al., 2011) and Miwi (Simon et al., 2011) (Figures S7A–S7C).
The Y350A/Y382A and F370A/Y379A mutations substantiallyreduced the piRNA loading (Figure 4D), confirming the importance of the recognition of the 20 -O-methylated piRNA 30 end
in this process. This result is consistent with a previous observation that Siwi-bound piRNAs are stabilized by Hen1-mediated20 -O-methylation (Izumi et al., 2016). Tyr379 of Siwi is conserved
as a tyrosine or a phenylalanine among the PIWIs (Phe342 of
Hiwi1 and Phe343 of Miwi) (Figure S5), and contributes tocreating the spacious pocket for the 20 -O-methyl group (Figures
4C and S7A–S7C). hAgo2 has a restricted 30 -nucleotide-binding
pocket (Figure 4E and S7D), as compared to those of the PIWIs,
since Tyr379 of Siwi is replaced with a smaller valine residue in
the AGOs (Val308 of hAgo2) (Figure S5). Together, these findings
highlight the functional importance of the spacious 30 -nucleotide-binding pocket of the PIWI-clade PAZ domains.
Potential t1A-Binding Pocket
Eukaryotic AGO proteins, such as hAgo2 (Lewis et al., 2005), anda subset of PIWI proteins, such as Siwi and Drosophila Aubergine (Wang et al., 2014), prefer target RNAs with an adenine at
the position opposite the first nucleotide in their guide RNAs
(referred to as t1A). The guide-target-bound hAgo2 structures revealed that the t1A nucleotide is accommodated within a binding
pocket at the L2-MID interface (Schirle et al., 2015) (Figure 5A).
The N6 of t1A forms a hydrogen bond with the side chain of
Ser561, whereas the N1 and N6 atoms of t1A form a water-mediated hydrogen-bonding network with Met437, Asn439, Lys440,
and Ile477. In addition, the t1A nucleobase forms a stacking
interaction with the side chain of Arg438.
The present structure revealed that Siwi has a potential
t1A-binding pocket similar to that of hAgo2 (Figure 5B). However, in the Siwi structure, the t1A-binding pocket is not fully
accessible, as the side chain of Arg520, equivalent to Arg438
in hAgo2, adopts a distinct conformation (Figure 5B). It is

B

A

Siwi

Siwi

PAZ

Y379

Y379

Y382

Y382
Y417 Y350

Y417

F370

F370

Y350

K386

Y383
Y387

28

27

K386

Y383
Y387

28

28

26
Y355

Y355
27

27

26

C

D

E
hAgo2

W
T
Y3
50
F3 A/Y
70 38
A/ 2A
Y3
79
A

Y379

Y417

26

F370

(nt)
100 90 80 70 -

V308
F312
H316

Y338

Y311

F294

60 -

R315

50 21

40 Um2830 -20- piRNA
19- FLAG-Siwi

Figure 4. Recognition of the piRNA 30 Segment(A) Binding of the piRNA 30 end to the Siwi-PAZ domain.
(B) Recognition of the piRNA 30 segment by the Siwi-PAZ domain (stereoview). Hydrogen bonding and electrostatic interactions are indicated by cyan dashedlines.
(C) Recognition of the 20 -O-methylated piRNA 30 end. The 20 -O-methyl group is indicated by a red arrow.
(D) piRNA loading in the wild-type and mutants of Siwi. FLAG-Siwi was used as a loading control.
(E) Recognition of the miRNA 30 segment by the hAgo2-PAZ domain (PDB: 4W5N).
See also Figure S7 and Table S2.

thus likely that Arg520 undergoes a conformational change toaccommodate the t1A nucleotide. Ser561 of hAgo2 is replacedwith a threonine in Siwi (Thr640) and Drosophila Aubergine,whereas it is replaced with a bulkier glutamine in BmAgo3 andDrosophila Ago3 (Figure S5), suggesting that the t1A binding
pocket is occluded by the glutamine residues in BmAgo3 andDrosophila Ago3, which lack the t1A preference.
Catalytic Tetrad in the PIWI Domain
The crystal structures of the eukaryotic and prokaryotic AGOs
revealed that the PIWI domains adopt an RNase H fold, with
the DEDX catalytic tetrad (X is generally Asp or His) in the active
site (Figure 6A–6D). The catalytic tetrad was originally discovered in the KpAgo structure, in which the DEDD tetrad is formedby the DDD triad (Asp974, Asp1046, and Asp1198) and the gluta-

mate finger (Glu1013) on a loop region (referred to as PIWI
domain loop 2) between the third b strand and the first a-helix
in the RNase H fold (Nakanishi et al., 2012) (Figure 6A). In
the hAgo2 structure, the DEDH tetrad is formed by the DDH triad(Asp597, Asp669 and His807) and the glutamate finger (Glu637)(Schirle and MacRae, 2012; Elkayam et al., 2012) (Figure 6B).
hAgo1 also has a catalytic tetrad similar to that of hAgo2 (Faehnle et al., 2013; Nakanishi et al., 2013). In the guide-bound
TtAgo structure, PIWI domain loop 2 is disordered (the ‘‘unplugged’’ conformation), whereas in the guide-target boundstructure, the loop region becomes ordered and the glutamate
finger (Glu512) is inserted within the DDH triad to complete
the catalytic tetrad (the ‘‘plugged-in’’ conformation) (Shenget al., 2014) (Figures 6C and 6D). Thus, unlike the eukaryotic
AGOs, the guide-target base pairing induces the ‘‘unplugged’’
Cell 167, 484–497, October 6, 2016 491

7

A

8

6
9

hAgo2

PAZ
P
PA
A

1

5
5
4

4

2

3
3

MID
MID
D

2

5′

t1A
1A
1A

L1
L1
3′

561
61
S561

L2
2

N

t1A
1
1A

PIWI
PIW
W

477
77
7
I477

4
M437

D43
D4
43
36
D436

MID
K44
4
K440

R438

L2

N4
N439

B
1
5

Siwi

4
3

MID
D

PA
PAZ
AZ
A
Z

2

5′

L1
T64
T640

L2
L2
R520

N

M519
1

T555

MID

PW
PIW
PIWI
522
22
22
K522
D51
D518
S521

L2

Figure 5. t1A-Binding Pocket(A and B) t1A-binding pockets of hAgo2 (PDB: 4W5O) (A) and Siwi (B). Surface and ribbon representations of the t1A-binding pocket are shown in the left and rightpanels, respectively. Water molecules are shown as red spheres. Hydrogen bonds are indicated by cyan dashed lines.

to ‘‘plugged-in’’ conformational changes in TtAgo. In these eukaryotic and prokaryotic AGO structures, the plugged-in conformation is stabilized through a conserved hydrogen-bonding
network within the PIWI domain (Figure 6A–6C).
The present structure provides the first structural insight into
the PIWI domain of PIWI-clade Argonautes and reveals that
the PIWI domain of Siwi adopts an RNase H fold similar to thoseof the AGO-clade Argonautes (Figure 6E). In addition, the residues involved in the formation of the catalytic tetrad (Asp670,
Glu708, Asp740, and His874 in Siwi) and the hydrogen-bonding
network (His673, Ser681, Arg739, and Glu754 in Siwi) areconserved among the PIWI-clade Argonautes (Figure S5).
Indeed, like the D670A, D740A, and H874A mutations, theE708A mutation of Siwi abolished the slicer activity (Figure 6F),
indicating that Glu708 participates in the catalytic-tetrad formation. However, in the present Siwi structure, PIWI domain loop 2(residues 704–708) is disordered, and thus Glu708 adopts theunplugged state, although the other active-site residues arelocated at positions similar to those of hAgo2 (Figure 6E). These
492 Cell 167, 484–497, October 6, 2016

observations suggest that, as in TtAgo, the catalytic tetrad of
Siwi becomes completed via a conformational change following
guide-target base pairing.
DISCUSSION
In this study, we affinity-purified the endogenous Siwi-piRISC
from BmN4 cells and determined its crystal structure at 2.4 Å
resolution. The Siwi-piRISC structure reveals the mechanism
by which Siwi recognizes the g1U and the 20 -O-methylated
30 end in the piRNA guide. Our structural and mutational analyses
further demonstrate that the catalytic tetrad in the PIWI domain
is critical for the slicer activity of Siwi, thereby suggesting thatthe catalytic tetrad is a conserved feature among the AGOand PIWI-clade Argonautes. Unlike the guide-bound eukaryotic
AGO structures, the Siwi-piRISC structure adopts the unpluggedconformation, as in the guide-bound TtAgo structure. The present structure also reveals that, unexpectedly, Siwi recognizes
the 50 phosphate of the piRNA in a metal-dependent manner,

A

B

KpAgo (plugged-in)
PL2
E1013

C

hAgo2 (plugged-in)

PL1

PL2

PL1

PL2

S
610
S610

637
7
E637

S985
9

TtAgo (plugged-in)

R486
48

55
53
E553
H977

H600

E10
E1060
10
106
1
0 0

E683
68
68
683
D97
74
7
4
D974

597
97
7
D597

R1
R104
R10
10 5
104
R1045

R668
R6
6
D66
D6
6
669
D669

D
D54
54
546
4
D546

D1
D119
D
119
1 98
D1198

80
807
07
07
H807

PL2

PL2

E708A

H874A

D740A

D670A

F

Siwi (unplugged)

WT

D660
D
660
660
0

Input

E

TtAgo (unplugged)
E512

D478
78
7
8

R545

D
D10
D1
D104
1
10
046
D1046

D

PL1

E512

PL1

- Target RNA (64 nt)

E553
553

R486
R4
R48
486
4
6 PL1
S681
E754
E7
E754
D4
D
47
47
478
78
8
D478

R545
D54
D54
4
D546

H673

- 3′ Cleavage product

D670
70
7
0

R739
D740
D74
D
74
74

D6
D
660
0
D660

H8
87
74
4
H874

- 5′ Cleavage product- FLAG-Siwi- piRNA

Figure 6. Catalytic Tetrad(A–E) Catalytic tetrads in the PIWI domains of KpAgo (PDB: 4F1N) (A), hAgo2 (PDB: 4W5N) (B), TtAgo (plugged-in) (PDB: 4NCB) (C), TtAgo (unplugged) (PDB:
4N47) (D), and Siwi (E). The DEDX catalytic tetrads and the surrounding residues are shown as yellow and green sticks, respectively. Hydrogen bonding andelectrostatic interactions are indicated by cyan dashed lines. The disordered regions are shown as dashed lines. PL, PIWI domain loop.
(F) Slicer activities of the wild-type and mutants of Siwi. Western blots for FLAG-Siwi and 32P-labeled piRNAs are shown below.
See also Table S2.

as observed in prokaryotic, but not in eukaryotic, AGOs. These
structural findings indicate that the eukaryotic PIWIs and theprokaryotic AGOs share unanticipated similarities in the catalytic-tetrad formation and the 50 -phosphate recognition. Most
notably, the present structure reveals unexpected differences
between Siwi and the eukaryotic AGOs in the arrangement of
the N-PAZ lobe relative to the MID-PIWI lobe. These structuraldifferences probably reflect the distinct lengths of their guideRNAs, as well as the mechanistic differences in RISC assemblyand cleavage product release.
The Siwi-piRISC primarily contains a 27–28-nt piRNA,
whereas the hAgo2-RISC contains a 21-nt miRNA (Xiol et al.,2014; Nishida et al., 2015). In the structure of hAgo2 bound toa 21-nt guide RNA (Schirle et al., 2014), the 50 and 30 ends ofthe guide are anchored within the binding pockets in the MIDPIWI and PAZ domains, respectively, whereas nucleotides
8–11 are disordered (Figure 7A). Nucleotides 2–7 (the seed region) adopt a helical conformation for base-pairing with thetarget RNA, whereas nucleotides 12–21 are bound to the
N-PAZ lobe, with nucleotides 14–18 threaded through a narrow
channel between the N and PAZ domains. In the present SiwipiRISC structure, nucleotides 1–5 adopt conformations similar
to those in the hAgo2-RISC structure (Figure 7B). In contrast,
the rest of the Siwi-bound piRNA adopts a conformation distinct

from that of the Ago2-bound miRNA, due to the structural differences in their N-PAZ channels. Importantly, the PAZ domain ofSiwi contains the PIWI-specific insertion (residues 405–412;
referred to as PIWI-Ins) (Figure S5), which protrudes into the
N-PAZ channel and may facilitate the binding of longer piRNAs(Figure 7B). Although the precise trajectory of the bound piRNA
is unknown, the structure of Siwi bound to a piRNA with a definedsequence may clarify the mechanism of piRNA recognition by
Siwi, as in the case of hAgo2 (Schirle et al., 2014).
Eukaryotic AGOs bind an 21-bp small RNA duplex and theneject the passenger strand (Czech and Hannon, 2011; Kobayashi and Tomari, 2016). In contrast, PIWIs are loaded with a single-stranded piRNA precursor, and its 30 end is then processedby nucleases, such as Zucchini, Trimmer, and Nibbler (Czech
and Hannon, 2016). In the human AGOs, the N domain (Kwakand Tomari, 2012; Schürmann et al., 2013) and the PAZ domain(Gu et al., 2012; Park and Shin, 2015) play important roles in the
RISC assembly process. Thus, the observed structural differences between the N-PAZ lobes of Siwi and hAgo2 likely reflect
their mechanistic differences in RISC assembly.
Cleavage of target RNAs by mammalian Ago2 (Jo et al., 2015;
Salomon et al., 2015) and Drosophila Ago2 (Yao et al., 2015;
Nishida et al., 2015) destabilizes the base pairing between theguide and target RNAs, thereby leading to the autonomous
Cell 167, 484–497, October 6, 2016 493

A

hAgo2
miRNA (21 nt)

B

Siwi
piRNA (~28 nt)

PA
PAZ
A
AZ
20
20

21
1

3′
19
1
9

MID

1

16
6

17
7

PA
PA
PAZ
AZ
Z

miRNA

MID
D

18
18

1
2

2

5′

1
12

4

13
1

3′

5′ 3

15
5

3

14

28
8
27
2
7

4

26 piRNA

5
5

7
6

N
L2

C

L1

L2
L

hAgo2
guide-target duplex(model)

D

L1

N

Siwi
guide-target duplex(model)

PAZ
PA
AZ
PAZ
PA
A
PAZ
MID
MID
D

MID
MID
D

3′

5′

5′

3′

PIW
PIWI
W

PIW
PIW
PIWI
W
N
L1
L1

N
L1
1

Figure 7. Nucleic-Acid-Binding Channel(A and B) Nucleic-acid-binding channels of hAgo2 (PDB: 4W5N) (A) and Siwi (B). The L0 and PIWI domains are omitted for clarity. A possible trajectory of thebound piRNA is shown as a dotted line.
(C and D) Structures of hAgo2 (PDB: 4W5N) (C) and Siwi (D) bound to a modeled guide-target duplex. In (B) and (D), the PIWI-Ins is highlighted in magenta.

release of cleavage products. In contrast, Siwi cannot release
the cleavage products autonomously and requires the DEADbox RNA helicase BmVasa for their efficient release (Nishida
et al., 2015). BmVasa associates with Siwi and BmAgo3 and facilitates the transfer of the 30 cleavage products from Siwi to
BmAgo3 in the ping-pong cycle (Xiol et al., 2014; Nishida et al.,2015). Modeling of the guide-target duplex into the hAgo2 structure indicates a steric clash between the modeled duplex and the
N domain (Figure 7C), suggesting that the widening of thenucleic-acid-binding channel is required for hAgo2 to accommodate an extensively paired guide-target duplex for target
cleavage, as previously proposed (Schirle et al., 2014). Thisoutward displacement of the N-PAZ lobe probably generates
494 Cell 167, 484–497, October 6, 2016an inward pressure in the hAgo2 protein, which may facilitate
the product release. In contrast, the modeling of the guide-target
duplex into the Siwi structure suggests that the N domain would
circumvent such severe clashes; instead, it would provide favorable interactions with the duplex (Figure 7D). These structuraldifferences might be the reasons why, after target cleavage,
the nicked target strand remains bound to Siwi quite stably. As
a consequence, Siwi requires BmVasa for efficient productrelease, in contrast to hAgo2, which releases the cleavage products autonomously. However, further structural and functional
studies are needed to verify this model.
In summary, the present findings have provided a framework
for understanding the mechanisms underlying piRISC-mediatedtransposon silencing. As in the case of hAgo2, the structures ofSiwi in complex with a piRNA and its target RNA provide mechanistic insights into target recognition and cleavage by the SiwipiRISC. Furthermore, the structures of Siwi-piRISC bound tointeractors, such as BmVasa, shed light on the mechanism bywhich Siwi and the interactors cooperatively function in the
ping-pong cycle. Importantly, the piRISC purification strategy
established here could be applicable to other PIWI proteins,when a specific antibody for an individual PIWI is available.
Accumulation of structural information about other PIWIs,
regardless of the species, will help to clarify the reasons why
the lengths of the mature piRNAs differ among PIWI proteinsfrom different species and why the g1U bias is not observed in
the piRNAs loaded onto a subset of PIWIs, such as BmAgo3.
Collectively, the present study paves the way for deciphering
the molecular mechanisms of piRISC-mediated transposon
silencing.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILSB Cell Lines
d METHOD DETAILS

Received: July 22, 2016
Revised: August 29, 2016
Accepted: August 31, 2016
Published: September 29, 2016
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B Small RNA Isolation from the

Science) from the Japan Agency for Medical Research and Development(AMED). H.N., K.M.N., H.S., and M.C.S. were supported by a Grant-in-Aid
for Scientific Research, from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (26291010 and 15H01463 to H.N.,
15K06948 to K.M.N., 25221003 to H.S., and 25221101 to M.C.S.). O.N. was
supported by the Core Research for Evolutional Science and Technology(CREST) Program, The Creation of Basic Medical Technologies to Clarify
and Control the Mechanisms Underlying Chronic Inflammation of Japan Science and Technology Agency (JST).

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SUPPLEMENTAL INFORMATION

Evans, P.R., and Murshudov, G.N. (2013). How good are my data and what is
the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214.

Supplemental Information includes seven figures and two tables and can befound with this article online at http://dx.doi.org/10.1016/j.cell.2016.09.002.

Faehnle, C.R., Elkayam, E., Haase, A.D., Hannon, G.J., and Joshua-Tor, L.
(2013). The making of a slicer: activation of human Argonaute-1. Cell Rep. 3,
1901–1909.

AUTHOR CONTRIBUTIONS

Feltzin, V.L., Khaladkar, M., Abe, M., Parisi, M., Hendriks, G.J., Kim, J., andBonini, N.M. (2015). The exonuclease Nibbler regulates age-associated traits
and modulates piRNA length in Drosophila. Aging Cell 14, 443–452.

N.M. purified and crystallized the complex and solved the structure. H.N.
conceived the purification strategy using a protease, collected the diffraction
data, and assisted with the structural analysis. K.S. and K.M.N. performed
the mutational analysis. T.H. and H.S. established the purification strategy
for endogenous PIWI-clade proteins. R.I. assisted with the structural analysis.
N.M., H.N., H.S., M.C.S., and O.N. designed the experiments and wrote themanuscript. H.N., M.C.S., and O.N. supervised all of the research.
ACKNOWLEDGMENTS
We thank Akira Oe for assistance with sample preparation, Takashi Yamano
for assistance with data collection, Dr. Takanori Nakane and Dr. Tomohiro
Nishizawa for assistance with model building, and the beamline scientists at
SPring-8 BL41XU for assistance with data collection. This research was supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life

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STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

Antibodies
Mouse monoclonal anti-Siwi antibody

Nishida et al., 2015

N/A

Dynabeads Protein G

Invitrogen

Cat# 10009D

Chemicals, Peptides, and Recombinant Proteins
CNBr-activated Sepharose 4 Fast Flow

GE Healthcare

Cat# 17098101

Thermolysin

Promega

Cat# V4001

Epicenter

Cat# AS3107

This paper

PDB: 5GUH

Kawaoka et al., 2009

N/A

pIB-3 3 FLAG-Siwi

Nishida et al., 2015

N/A

pIB-3 3 FLAG-Siwi, various mutants

This paper

N/A

Table S2

N/A

Critical Commercial Assays
T7 High Yield Transcription Kit
Deposited Data
Atomic coordinates, Siwi-piRISC
Experimental Models: Cell Lines
BmN4 cells
Recombinant DNA

Sequence-Based ReagentsPrimer sequences
Software and Algorithms
DIALS

Waterman et al., 2013

http://dials.lbl.gov

AIMLESS

Evans and Murshudov, 2013

http://www.ccp4.ac.uk/html/aimless.html

MOLREP

Vagin and Teplyakov, 2010

http://www.ccp4.ac.uk/html/molrep.html

Buccaneer

Cowtan, 2006

http://www.ysbl.york.ac.uk/cowtan/buccaneer/buccaneer.html

COOT

Emsley and Cowtan, 2004

http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot

PHENIX

Adams et al., 2010https://www.phenix-online.org

RESOLVE

Terwilliger, 2000

https://solve.lanl.gov/Resolve/resolve.html

CueMol

N/A

http://www.cuemol.org

Other
EX-CELL 420 Medium

Sigma-Aldrich

Cat# 14420C

FuGENE HD

Promega

Cat# E2311(CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding author Osamu Nureki(nureki@bs.s.u-tokyo.ac.jp).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell Lines
BmN4 cells were cultured at 27 C in EX-CELL 420 medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum (EquitechBio) and Penicillin-Streptomycin-Glutamine (Thermo Fisher).
METHOD DETAILS
Purification of the Siwi-piRISC
The anti-Siwi monoclonal antibody (30 mg) (Nishida et al., 2015) was coupled with CNBr-activated Sepharose 4 Fast Flow beads(12 ml) (GE Healthcare), according to the manufacturer’s instructions. BmN4 cells (9 3 108 cells) were suspended in buffer
e1 Cell 167, 484–497.e1–e2, October 6, 2016

30 mM Tris-HCl [pH 7.3], 300 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM DTT, 10% glycerol, 5 mg/ml aprotinin, 2 mg/ml leupeptin and
1 mg/ml pepstatin), lysed by a homogenizer, and then centrifuged. The supernatant was incubated with the anti-Siwi antibody-conjugated beads at 4 C for 4 hr, and the mixture was then loaded into an Econo-Column (Bio-Rad). The beads were washed three times
with buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 0.5 mM CaCl2, 0.1% NP-40, 1 mM DTT and 10% glycerol), and then incubatedwith thermolysin (2 mg) (Promega) at 4 C for 12 hr, to release the Siwi-piRISC core. The Siwi-piRISC core was loaded onto a HiTrap
Heparin HP column (GE Healthcare), equilibrated with buffer (50 mM Tris-HCl [pH 8.0], 200 mM NaCl, 1 mM DTT and 10% glycerol),and it was then eluted using a linear gradient of 0.2–2 M NaCl. The Siwi-piRISC core was further purified by chromatography on
a HiLoad 16/600 Superdex 200 column (GE Healthcare), equilibrated with buffer (10 mM Tris-HCl [pH 8.0], 300 mM NaCl, 1 mM
DTT and 10% glycerol).
Crystallization and Structural Determination
The purified Siwi-piRISC was crystallized at 20 C, using the sitting-drop vapor diffusion method. The crystallization drops wereformed by mixing 0.1 ml of Siwi-piRISC solution (A280 nm = 10–15) and 0.1 ml of reservoir solution (50 mM potassium phosphate monobasic and 20% PEG 8000). The crystals were cryoprotected by the reservoir solution supplemented with 20% glycerol. X-ray diffraction data were collected at 100 K on the beamline BL41XU at SPring-8 (Hyogo, Japan). The diffraction data were processed using
DIALS (Waterman et al., 2013) and AIMLESS (Evans and Murshudov, 2013). The initial phases were calculated by molecular replacement with MOLREP (Vagin and Teplyakov, 2010), using the hAgo2 structure (PDB: 4OLA) (Schirle and MacRae, 2012) as the searchmodel. The initial model was automatically built using Buccaneer (Cowtan, 2006), and then improved by manual model building using
COOT (Emsley and Cowtan, 2004) and structural refinement with PHENIX (Adams et al., 2010), resulting in a clear electron density for
the MID and PIWI domains. Subsequently, molecular replacement was performed with MOLREP, using the structure of the isolated
PAZ domain from the Hiwi1 structure (PDB: 3O7V) (Tian et al., 2011) as the search model. The electron density map was improved bydensity modification with RESOLVE (Terwilliger, 2000), and the model was iteratively built and refined using COOT and PHENIX,
respectively. The data collection and refinement statistics are summarized in Table S1. All molecular graphics were prepared with
CueMol (http://www.cuemol.org).
Small RNA Isolation from the Immunopurified Siwi Complex
Immunoprecipitation was performed essentially as described previously (Saito et al., 2006). BmN4 lysates were prepared in binding
buffer (30 mM HEPES [pH 7.4], 150 mM potassium acetate, 5 mM magnesium acetate, 5 mM DTT, 0.1% Tergitol-type NP-40, 5 mg/ml
aprotinin, 2 mg/ml leupeptin and 2 mg/ml pepstatin), and then incubated with an anti-Siwi antibody bound to Dynabeads Protein G(Invitrogen) in the presence of 500 mM NaCl. The beads were then washed three times with binding buffer supplemented with
500 mM NaCl, and twice with binding buffer. Total RNAs were eluted from the beads by phenol-chloroform treatment, and thenprecipitated with ethanol. The RNAs were dephosphorylated with Antarctic Phosphatase (NEB) and radiolabeled with 32P-g-ATP
using T4 PNK (NEB).
Plasmid Construction
The expression vector for FLAG-tagged Siwi was generated by Gateway Technology (Invitrogen), using the pIB-3 3 FLAG vector. The
expression vectors for the FLAG-Siwi mutants were generated by inverse PCR, using the wild-type FLAG-Siwi vector as the template.
The sequences of the DNA oligos used for inverse PCR are listed in Table S2.
In Vitro Target RNA Cleavage Assay
Cleavage assays were performed as described previously (Miyoshi et al., 2005). The sequence of the RNA substrate used is
50 -GGGAGUCCAGAUUUGAAUCCGUUAGAUUACAAUCAAGCUUAUCGAUACCGUCGACCUCGAGGGG-3 0 (64 nt), which corresponds to part of the Bmmar6 transposon mRNA. The RNA substrate was prepared by in vitro transcription using a DNA template,which was amplified by PCR using the two DNA oligos, Target-F and Target-R (Table S2). In vitro transcription was performed with a
T7 High Yield Transcription Kit (Epicenter) in the presence of 32P-a-UTP.
QUANTIFICATION AND STATISTICAL ANALYSISRNA cleavage and piRNA loading experiments were repeated three times, and representative results were shown.
DATA AND SOFTWARE AVAILABILITY
Data Resources
The atomic coordinates of the Siwi-piRISC have been deposited in the Protein Data Bank: 5GUH.

Cell 167, 484–497.e1–e2, October 6, 2016 e2

Supplemental Figures
A

Full-length Siwi-piRISC

Siwi-piRISC core

Siwi

N-terminal disordered region

Siwi

Siwi

Siwi

piRNA
Thermolysin

䌦

䌦

+

Thermolysin

100
75
50
37

280 nm
260 nm

300

250
150
- Siwi (FL)
- Siwi (core)- HC- Thermolysin

Absorbance (mAU)

(kDa)

C

Sup

B

Beads

anti-Siwi antibody beads

D

E

(kDa)

(nt)
100 90 80 70 -

250
150
100
75

Siwi (core)

60 50 -

150

50
40 37
30 -

25
20

- LC

0

0

20

40

60

80

Elution volume (ml)

100

120

piRNA

25
20
20 -

Figure S1. Preparation of the Siwi-piRISC, Related to Figure 1(A) Purification scheme of the endogenous Siwi-piRISC from BmN4 cells.
(B) Separation of the Siwi-piRISC core from the anti-Siwi antibody beads. The full-length (FL) Siwi-piRISC bound to the antibody beads was incubated in theabsence and presence of thermolysin at 4 C overnight. The untreated beads and the supernatants were then analyzed by SDS-PAGE. The gel was stained with
SimplyBlue SafeStain (Invitrogen). HC, heavy chain; LC, light chain.
(C) Elution profile of the purified Siwi-piRISC core from the HiLoad 16/600 Superdex 200 column.
(D) SDS-PAGE analysis of the purified Siwi-piRISC core. The gel was stained with SimplyBlue SafeStain.
(E) Denaturing urea-PAGE analysis of the purified Siwi-piRISC core. The bound piRNA was 50 32P-end labeled.

A

5′
3′

B

C

U1

3′

28

5′
27

26
2
3
4
5

Figure S2. Electron Density Map, Related to Figure 1(A) mFO  DFC omit electron density map for the bound piRNA (green mesh, contoured at 3.5s).
(B) 2mFO  DFC electron density map for the piRNA 50 segment (gray mesh, contoured at 1s).
(C) 2mFO  DFC electron density map for the piRNA 30 segment (gray mesh, contoured at 1s).
The mFO  DFC omit electron density map for the 20 -O-methyl group is depicted as a green mesh (contoured at 2.2s).

A
L0

N

RMSD = 0.88 Å (11 Cα)
SEQ ID = 28%

RMSD = 1.7 Å (66 Cα)
SEQ ID = 27%

L1

PAZ

RMSD = 2.0 Å (94 Cα)
SEQ ID = 21%
RMSD = 1.9 Å (69 Cα)
SEQ ID = 13%

B

L2

MID

PIWI

RMSD = 2.0 Å (89 Cα)
SEQ ID = 17%

RMSD = 1.6 Å (125 Cα)
SEQ ID = 19%

RMSD = 1.7 Å (224 Cα)
SEQ ID = 32%

PAZ

L1

N

Figure S3. Structural Comparison between the Individual Domains of Siwi and hAgo2, Related to Figure 2(A) Superimpositions of the individual domains of Siwi (colored) and hAgo2 (PDB: 4W5N) (beige). Sequence identities and root mean square deviation (RMSD)
values for equivalent Ca atoms are shown below the structures.
(B) Superimpositions of the N-PAZ lobe of Siwi (colored) and hAgo2 (PDB: 4W5N) (beige), based on their N domains.

Siwi
hAgo2

1 MSEPRGRGRARGRAGRGGDGGGPAPRRPGEQAGPSQQSMPPGPRPQPPSGWGPQSSVPPVRAGVPTPTAQAGRASHRVTPTTHEHPGDIDVQQRM Q............................................................................................... .
Core region (residues 130–899)
β1

Siwi
hAgo2

β2

β1
β4
Siwi
hAgo2

β5

β4
β9

η1

β5

β9

β11

β12
β16

β15

α4

α4

α5

β16

α8

β13

α5

β17

β18

PIWI-Ins

η3

β13

β19

α6

α6
α9

β17
β20

β21

β18 η1
β22

η4

β23

β19
α10

436 F K L M R S L D V H T K I G P D K R I E K L N N F N R R F T S T P E V V E E L A T W S L K L S K E L V K I K G R Q L P P E N I I Q A N N . . V K Y P A G D T T E G W T R D M R S K H L L A I A Q
358 D N Q T S T M I R A T A R S A P D R Q E E I S K L M R S A S F N T D . . P Y V R E F G I M V K D E M T D V T G R V L Q P P S I L Y G G R N K A I A T P V Q G V W . . . . D M R N K Q F H T G I E
α7

α8

η2

α9

α11

β20

β21

β25

β22

β23
Specificity Loop
β26

α12

α13

530 L N S W V V I T P . . . . E R Q R R D T E S F I D L I I K T G G G V G F R M R S P D L V V I R H D G P I E Y A N M C E E V I A R K . N P A L I L C V L A R N Y A D R Y E A I K K K C T V D R A V
448 I K V W A I A C F A P Q R Q C T E V H L K S F T E Q L R K I S R D A G M P I Q G Q P C F C K Y A Q G A D S V E P M F R H L K N T Y A G L Q L V V V I L P . G K T P V Y A E V K R V G D T V L G M
β24
β27

α10
α14

β25

α15

η3

β28

α11
β29

β26

α12

β30

β31

621 P T Q V V C A R N M S S K S A M S I A T K V A I Q I N C K L G G S P W T V D . . . . . . I P L P S L M V V G Y D V C H D T R . . S K E K S F G A F V A T L D K Q M T Q Y Y S I V N A H T S G E E
543 A T Q C V Q M K N V Q R T . T P Q T L S N L C L K I N V K L G G V N N I L L P Q G R P P V F Q Q P V I F L G A D V T H P P A G D G K K P S I A A V V G S M D A H P N R Y C A T V R V Q Q H R Q E
β27

α13

α14

β28

α16

β32

η4

η5

β29

β30

α17

β33

β31
β34

β35

709 L S S H M G F N I A S A V K K F R E K N G T Y P A R I F I Y R D G V G D G Q I P Y V H S H E V A E I K K K L A E I Y A G V E I K L A F I I V S K R I N T R I F V Q R G . . . . . R S G E N P R P
638 I I Q D L A A M V R E L L I Q F Y K S T R F K P T R I I F Y R D G V S E G Q F Q Q V L H H E L L A I R E A C I K L E K D Y Q P G I T F I V V Q K R H H T R L F C T D K N E R V G K S G . N I P A
α15
β36

Siwi
hAgo2

α3

α3

349 D Y N . . . K R T Y R V D D V A W N V S P K S T F K M R D . . . . . E N I T Y I E Y Y Y K K Y N L R I Q D P G Q P L L I S R S K P R E I R A G L P E L I Y L V P E L C R Q T . G L S D E M R A N
270 T H C G Q M K R K Y R V C N V T R R P A S H Q T F P L Q Q E S G Q T V E C T V A Q Y F K D R H K L V L R Y P H L P C L Q V G Q E . . . . . . . . Q K H T Y L P L E V C N I V A G Q R C I K K L T

β24

Siwi
hAgo2

α1
α2

α2

β12

β15

β14

Siwi
hAgo2

β3
AGO-Ins

β7

β11

β10

α7

Siwi
hAgo2

β2

β7

β6

β10 η2

β14

Siwi
hAgo2

β6

264 L Q L M G R D Y F D P E A K I D I P E F . K L Q I W P G Y K T T I N Q Y E D R L L L V T E I A H K V L R M D T . V L Q M L S E Y A A T K . . . . . . . . . G N N Y K K I F L E D V V G K I V M T
174 Y T P V G R S F F T A S E G C S N P L G G G R E V W F G F H Q S V R P S L W K M M L N I D V S A T A F Y K A Q P V I E F V C E V L D F K S I E E Q Q K P L T D S Q R V K F T K E I K G L K V E I
β8

Siwi
hAgo2

α1

193 S K T L G G . . . . . . . Y L F D G T . V L Y T V N R L H . . P D P M E L Y S D . . R K T D N E R M R I L I K L T C E V . . . . . . . . . . . . . S P G D Y H Y I Q I F N I I I R K C F N L L K
79 V Q H F K T Q I F G D R K P V F D G R K N L Y T A M P L P I G R D K V E L E V T L P G E G K D R I F K V S I K W V S C V S L Q A L H D A L S G R L P S V P F E T I Q A L D V V M R H L P S M R .

β8
Siwi
hAgo2

β3

97 K L E L G P H S S G G G D A S S V V G R G S R R G G G R V L P E T I S I L R T R P E A V T S K K G T S G T P L D L L A N Y F T V E T T P K W G L Y Q Y H V D I S P E E D S T G V R K A L M R V H
1 .................MYSGAGPALAPPAPPPPIQGYAFKPPPRPDFGTSGRTIKLQANFFEMD.IPKIDIYHYELDIKPEKCPRRVNREIVEH M

β32
β37

η5
β38

α16

β33

α18

β34
α19

800 G T V I D D V V T L P E R Y D F Y L V S Q N V R E G T I A P T S Y N V I E D T T G L N P D R I Q R L T Y K L T H L Y F N C S S Q V R V P S V C Q Y A H K L A F L A A N S L H N Q P . . . . . . .
733 G T T V D T K I T H P T E F D F Y L C S H A G I Q G T S R P S H Y H V L W D D N R F S S D E L Q I L T Y Q L C H T Y V R C T R S V S I P A P A Y Y A H L V A F R A R Y H L V D K E H D S A E G S
β35

β36

β37

α17

η6

α18

η6
Siwi
hAgo2

889 . . . . . . . . . . . . . . . . . . . . H Y S L N E T L Y F L
829 H T S G Q S N G R D H Q A L A K A V Q V H Q D T L R T M Y F A
α19

η7

N-L1-L2 interface

L1-PAZ-L2 interface

5′-phosphate recognition

Metal binding

3′-segment recognition

t1A-binding pocket

Catalytic tetrad

H-bond network in the active site

Seed recognition

Figure S4. Structure-Guided Sequence Alignment of Siwi and of hAgo2, Related to Figure 2
The structures of Siwi and hAgo2 (PDB: 4W5N) were superimposed by the secondary-structure matching (SSM) algorithm, using CueMol (http://www.cuemol.
org), and then the sequence alignment was manually refined. The AGO- and PIWI-specific insertions are highlighted in yellow and magenta, respectively. The
specificity loop is highlighted in orange. Key residues are marked by triangles.

β1
Siwi
BmAgo3
DmPiwi
DmAub
DmAgo3
Miwi
hAgo2
DmAgo1

130
155
76
90
107
92
17
127

β2

β3

α1

β4

β5

β6

ISILRTRPEAVTSKKGTSGTPLDLLANYFTVETTPKWGLYQYHVDISPEEDSTGVRKALMRVHSKTLG.......GYLFDGT.VLYTVNRLHPD....PMELYSDRKTDNE R
KYFREVKDTPPVVKKGETGVPIEVTCNYIYLNFKE.NIVFEYEVKFEPDQDYKHLRFKLLNEHIEHFK.......EKTFDGT.TLYVPHELPDAV.....RNLVSTNP.YD Q
YDYLNTRPAELVSKKGTDGVPVMLQTNFFRLKTKPEWRIVHYHVEFEPSIENPRVRMGVLSNHANLLG......SGYLFDGL.QLFTTRKFEQEI.....TVLSGKSKLDI E
TDLVYSRPPGMTSKKGVVGTHITVQANYFKVLKRPNWTIYQYRVDFTPDVEATRLRRSFLYEHKGILG.......GYIFDGT.NMFCINQFKAVQDSPYVLELVTKSRAGE N
AIASENGLFFPDLIYGSKGSSVNIYCNYLKLTTDESKGVFNYEVRFFPPIDSVHLRIKYLNDHKDKLG......GTKTFDGN.TLYLPILLPNKM.....TVFISKA...E D
VNTRQNLDHVKESKTGSSGIIVKLSTNHFRLTSRPQWALYQYHIDYNPLMEARRLRSALLFQHEDLIG......RCHAFDGT.ILFLPKRLQHKV.....TEVFSQTRNGE H
PIQGYAFKPPPRPDFGTSGRTIKLQANFFEMDI.PKIDIYHYELDIKPEKCPRRVNREIVEHMVQHFKTQIFGDRKPVFDGRKNLYTAMPLPIGRDKVELEVTLPGEGKDR I
QPDMPVFTCPRRPNLGREGRPIVLRANHFQVTM.PRGYVHHYDINIQPDKCPRKVNREIIETMVHAY.SKIFGVLKPVFDGRNNLYTRDPLPIGNERLELEVTLPGEGKDR I
β7

α2

β8

β9

η1

β10 η2

β11

β12

α3

AGO-Ins
Siwi
BmAgo3
DmPiwi
DmAub
DmAgo3
Miwi
hAgo2
DmAgo1

230
252
176
194
204
192
128
237

MRILIKLTCEV.............SPGDYHYIQIFNIIIRKCFNLLKLQLMGRDYFDPEAKIDIPEF.KLQIWPGYKTTINQYEDRLLLVTEIAHKVLRMDT.VLQMLSEY A
SKVNVSIIFRR.............TRRLSEMIHIYNVMFKCIMKDLKLIRFGRQHYNEHAAIQIPQH.KLEVWPGYVTAVDEYEGGLMLTLDSTHRVLRTQT.VLSLIKEV VYKISIKFVGFI.............SCAEPRFLQVLNLILRRSMKGLNLELVGRNLFDPRAKIEIREF.KMELWPGYETSIRQHEKDILLGTEITHKVMRTET.IYDIMRRC S
IEIKIKAVGSV.............QSTDAEQFQVLNLILRRAMEGLDLKLVSRYYYDPQAKINLENF.RMQLWPGYQTSIRQHENDILLCSEICHKVMRTET.LYNILSDA I
VELQIRILYKK.............KEEMRNCTQLYNILFDRVMKVLNYVKFDRKQFDPSRPKIIPLA.KLEVWPGYVTAVDEYKGGLMLCCDVSHRILCQKT.VLEMLVDL YVRITITLTNEL.............PPTSPTCLQFYNIIFRRLLKIMNLQQIGRNYYNPSDPIDIPNH.RLVIWPGFTTSILQYENNIMLCTDVSHKVLRSET.VLDFMFNL YFKVSIKWVSCVSLQALHDALSGRLPSVPFETIQALDVVMR.HLPSMRYTPVGRSFFTASEGCSNPLGGGREVWFGFHQSVRPSLWKMMLNIDVSATAFYKAQPVIEFVCEV L
FRVTIKWQAQVSLFNLEEALEGRTRQIPYDAILALDVVMR.HLPSMTYTPVGRSFFSSPEGYYHPLGGGREVWFGFHQSVRPSQWKMMLNIDVSATAFYKAQPVIDFMCEV L
α4

β13

β14

β15

β16

α5

β17

β18
PIWI-Ins

Siwi
BmAgo3
DmPiwi
DmAub
DmAgo3
Miwi
hAgo2
DmAgo1

327
349
273
291
301
289
239
348

ATKGN.........NYKKIFLEDVVGKIVMTDYN...KRTYRVDDVAWNVSPKSTFKMR.D....ENITYIEYYYKKYNLRIQDPGQPLLISRSKPREIRAG.L..PELIY LQTEGA.........NWKRKMTDILIGASVMTTYN...KKLFRVDTIDDKMSPRSTFEKT.EKGETVQISFIDYYKKNYGIEIMDWDQPLLISRDTK.RMPGSDTPTDFMIC L.HNPA.........RHQDEVRVNVLDLIVLTDYN...NRTYRINDVDFGQTPKSTFSCK.G....RDISFVEYYLTKYNIRIRDHNQPLLISKNRDKALKTN.A..SELVV L.RDSD.........DYQSTFKRAVMGMVILTDYN...NKTYRIDDVDFQSTPLCKFKTN.D....GEISYVDYYKKRYNIIIRDLKQPLVMSRPTDKNIRGG.N..DQAIM I
QQNVE.........HYQESARKMLVGNIVLTRYN...NRTYKINDICFDQNPTCQFEIK.T....GCTSYVEYYKQYHNINIKDVNQPLIYSIKKS.RGI.PAERENLQFC LQQTEEH........KFQEQVSKELIGLIVLTKYN...NKTYRVDDIDWDQNPKSTFKKA.DG...SEVSFLEYYRKQYNQEITDLKQPVLVSQPKRRRGPGGTL..PGPAM LDFKSIEEQQKPLTDSQRVKFTKEIKGLKVEITHCGQMKRKYRVCNVTRRPASHQTFPLQQESGQTVECTVAQYFKDRHKLVLRYPHLPCLQVGQE...........QKHTY L
DIRDINEQRKPLTDSQRVKFTKEIKGLKIEITHCGQMRRKYRVCNVTRRPAQMQSFPLQLENGQTVECTVAKYFLDKYRMKLRYPHLPCLQVGQE...........HKHTY Lη3Siwi
BmAgo3
DmPiwi
DmAub
DmAgo3
Miwi
hAgo2
DmAgo1

419
447
364
382
394
384
340
449

β19

α6

α7

α8

α9

β20

β21

β22

η4

β23

VPELCRQT.GLSDEMRANFKLMRSLDVHTKIGPDKRIEKLNNFNRRFTSTPEVVEELATWSLKLSKELVKIKGRQLPPENIIQANNVKYP...............AGDTTE G
IPELCQLT.GLTDDQRSNFRLMKDVATYTRITPNQRHAAFKKYIESVMKNETAKSRLAGWGLSIAPETVNLTARTLPPETLYFGDNVRV................PGKPNA E
IPELCRVT.GLNAEMRSNFQLMRAMSSYTRMNPKQRTDRLRAFNHRLQNTPESVKVLRDWNMELDKNVTEVQGRIIGQQNIVFHNG.KVP................AGENA D
IPELARAT.GMTDAMRADFRTLRAMSEHTRLNPDRRIERLRMFNKRLKSCKQSVETLKSWNIELDSALVEIPARVLPPEKILFGNQ.KIF...............VCDARA D
IPELCYLT.GLRDEVRSDNKLMREIATFTRVSPNQRQMALNKFYENVSNTPAAQEILNSWGLSLTNNSNKISGRQMDIEQIYFSKISVS................AG.RSA E
IPELCYLT.GLTDKMRNDFNVMKDLAVHTRLTPEQRQREVGRLIDYIHKDDNVQRELRDWGLSFDSNLLSFSGRILQSEKIHQGGKTFD................YNPQFA DPLEVCNIVAGQRCIKKLTDNQTSTMIRATARSAPDRQEEISKLMRSASF..NTDPYVREFGIMVKDEMTDVTGRVLQPPSILYGGRNK...............AIATPVQG V
PLEVCNIVAGQRCIKKLTDMQTSTMIKATARSAPDREREINNLVKRADF..NNDSYVQEFGLTISNSMMEVRGRVLPPPKLQYGGRVSTGLTGQQLFPPQNKVSLASPNQG V
α10

β24

α11

β25

α12

β26

α13

Specificity Loop
Siwi
BmAgo3
DmPiwi
DmAub
DmAgo3
Miwi
hAgo2
DmAgo1

515
542
458
477
488
479
435
559

WTRDMRSKHLLAI..AQLNSWVVITPERQ....RRDTESFIDLIIKTGGGVGFRMRSPDLVVIRHDGPIEYANMCEEVIARK.NPALILCVLARNYADRYEAIKKKCTVDR AWNSEVTKHSVMQA..VDIMRWVLLFTQRD....KQVAMDFLSTLKRNCRPMGIMVSDAELVPLANDRTDTYVLALKK.CITS.SVQLVVAICSTKRDDRYAAIKKVCCADN PWQRHFRDQRMLTTPSDGLDRWAVIAPQRN....SHELRTLLDSLYRAASGMGLRIRSPQEFIIYDDRTGTYVRAMDD.CVRS.DPKLILCLVPNDNAERYSSIKKRGYVDR A
WTNEFRTCSMFKN..VHINRWYVITPSRN....LRETQEFVQMCIRTASSMKMNICNPIYEEIPDDRNGTYSQAIDN.AAAN.DPQIVMVVMRSPNEEKYSCIKKRTCVDR P
FSKHAVTNEMLKV..VHLSKWIIIHLRNY....RQAATSLLDNMKQACESLGMNISNPTMISLDHDRIDAYIQALRR.NITM.NTQMVVCICHNRRDDRYAAIKKICCSEI P
WSKETRGAPLISV..KPLDNWLLIYTRRN....YEAANSLIQNLFKVTPAMGIQMKKAIMIEV.DDRTEAYLRALQQ.KVTS.DTQIVVCLLSSNRKDKYDAIKKYLCTDC P
W..DMRNKQFHTG..IEIKVWAIACFAPQRQCTEVHLKSFTEQLRKISRDAGMPIQGQPCFCKYAQGADSVEPMFRHLKNTYAGLQLVVVILP.GKTPVYAEVKRVGDTVL G
W..DMRGKQFFTG..VEIRIWAIACFAPQRTVREDALRNFTQQLQKISNDAGMPIIGQPCFCKYATGPDQVEPMFRYLKITFPGLQLVVVVLP.GKTPVYAEVKRVGDTVL G

Siwi
BmAgo3
DmPiwi
DmAub
DmAgo3
Miwi
hAgo2
DmAgo1

620
646
564
581
592
582
542
666

VPTQVVCARNMS.....SKSAMSIATKVAIQINCKLGGSPWTVD......IPLPSLMVVGYDVCHDTRS..KEKSFGAFVATLDKQ.MTQYYSIVNAHTSGEELSSHMGFN I
VPSQVINARTLM....NTNKIRSITQKILLQLNCKLGGTLWSIS......IPFKSAMIVGIDSYHDPSR..RNRSVCSFVASYNQS.MTLWYSKVIFQEKGQEIVDGLKCC L
VPTQVVTLKTT.....KNRSLMSIATKIAIQLNCKLGYTPWMIE......LPLSGLMTIGFDIAKSTRD..RKRAYGALIASMDLQQNSTYFSTVTECSAFDVLANTLWPM I
VPSQVVTLKVIAPRQQKPTGLMSIATKVVIQMNAKLMGAPWQVV......IPLHGLMTVGFDVCHSPKN..KNKSYGAFVATMDQKESFRYFSTVNEHIKGQELSEQMSVN M
IPSQVINAKTLQ....NDLKIRSVVQKIVLQMNCKLGGSLWTVK......IPFKNVMICGIDSYHDPSN..RGNSVAAFVASINSS.YSQWYSKAVVQTKREEIVNGLSAS FTPSQCVVARTLG....KQQTVMAIATKIALQMNCKMGGELWRVD......MPLKLAMIVGIDCYHDTTA..GRRSIAGFVASINEG.MTRWFSRCVFQDRGQELVDGLKVC L
MATQCVQMKNVQRT......TPQTLSNLCLKINVKLGGVNNILLPQGRPPVFQQPVIFLGADVTHPPAGDGKKPSIAAVVGSMDAH.PNRYCATVRVQQHRQEIIQDLAAM V
MATQCVQAKNVNKT......SPQTLSNLCLKINVKLGGINSILVPSIRPKVFNEPVIFLGADVTHPPAGDNKKPSIAAVVGSMDAH.PSRYAATVRVQQHRQEIIQELSSM V

β27

α14

α15

α16

β32

η5

β28

β29

α17

β30

β33

β34

β31

β35

β36

β37

Siwi
BmAgo3
DmPiwi
DmAub
DmAgo3
Miwi
hAgo2
DmAgo1

718
745
663
685
691
681
647
771

ASAVKKFREKN.GTYPARIFIYRDGVGDGQIPYVHSHEVAEIKKKLAEIYAGVE....IKLAFIIVSKRINTRIFVQRG....RSGENPRPGTVIDDVVTLPERYDFYLVS Q
VDALTHYLRSN.GQLPDRIIIYRDGVGDGQLKLLQQYEIPQMKICFTIL....GSNYQPTLTYVVVQKRINTRIFLKSR....DGYDNPNPGTVVDHCITRRDWYDFLIVS QAKALRQYQHEH.RKLPSRIVFYRDGVSSGSLKQLFEFEVKDIIEKLKTEYARVQ.LSPPQLAYIVVTRSMNTRFFL........NGQNPPPGTIVDDVITLPERYDFYLVS QACALRSYQEQH.RSLPERILFFRDGVGDGQLYQVVNSEVNTLKDRLDEIYKSAGKQEGCRMTFIIVSKRINSRYFT........GHRNPVPGTVVDDVITLPERYDFFLVS Q
EIALKMYRKRN.GKLPTNIIIYRDGIGDGQLYTCLNYEIPQFEMVC.........GNRIKISYIVVQKRINTRIFSGSG....IHLENPLPGTVVDQHITKSNMYDFFLVS QQAALRAWSGCN.EYMPSRVIVYRDGVGDGQLKTLVNYEVPQFLDCLKSV....GRGYNPRLTVIVVKKRVNARFFAQSG....GRLQNPLPGTVIDVEVTRPEWYDFFIVS Q
RELLIQFYKST.RFKPTRIIFYRDGVSEGQFQQVLHHELLAIREACIKL....EKDYQPGITFIVVQKRHHTRLFCTDKNERVGKSGNIPAGTTVDTKITHPTEFDFYLCS HRELLIMFYKSTGGYKPHRIILYRDGVSEGQFPHVLQHELTAIREACIKL....EPEYRPGITFIVVQKRHHTRLFCAEKKEQSGKSGNIPAGTTVDVGITHPTEFDFYLCS H

Siwi
BmAgo3
DmPiwi
DmAub
DmAgo3
Miwi
hAgo2
DmAgo1

821
848
765
788
789
784
754
879

NVREGTIAPTSYNVIEDTTGLNPDRIQRLTYKLTHLYFNCSSQVRVPSVCQYAHKLAFLAANSLHNQPHYSL...........................NETLYFL
KVTQGTVTPTHYVVVYDDSGITPDQCQRLTYKMCHLYYNWPGTVRVPAPCQYAHKLSYLVGQCVHAQPSDVL...........................VDKLFFLQVRQGTVSPTSYNVLYSSMGLSPEKMQKLTYKMCHLYYNWSGTTRVPAVCQYAKKLATLVGTNLHSIPQNAL...........................EKKFYYLAVRIGTVSPTSYNVISDNMGLNADKLQMLSYKMTHMYYNYSGTIRVPAVCHYAHKLAFLVAESINRAPSAGL...........................QNQLYFL
LVRQGTVTPTHYVVLRDDCNYGPDIIQKLSYKLCFLYYNWAGTVRIPACCMYAHKLAYLIGQSIQRDVAEAL...........................SEKLFYLAVRSGSVSPTHYNVIYDSSGLKPDHIQRLTYKLCHVYYNWPGVIRVPAPCQYAHKLAFLVGQSIHREPNLSL...........................SNRLYYL
AGIQGTSRPSHYHVLWDDNRFSSDELQILTYQLCHTYVRCTRSVSIPAPAYYAHLVAFRARYHLVDKEHDSAEGSHTSGQSNGRDHQALAKAVQVHQDTLRTMYFAQGIQGTSRPSHYHVLWDDNHFDSDELQCLTYQLCHTYVRCTRSVSIPAPAYYAHLVAFRARYHLVEKEHDSGEGSHQSGCSEDRTPGAMARAITVHADTKKVMYFA

β38

α18

α19

η6

N-L1-L2 interface

L1-PAZ-L2 interface

5′-phosphate recognition

Metal binding

3′-segment recognition

t1A-binding pocket

Catalytic tetrad

H-bond network in the active site

Seed recognition

Figure S5. Multiple Sequence Alignment of the Argonaute Proteins, Related to Figure 2
The secondary structure of Siwi is indicated above the sequences. Key residues are marked by triangles. The figure was prepared using Clustal Omega (http://
www.ebi.ac.uk/Tools/msa/clustalo) and ESPript3 (http://espript.ibcp.fr/ESPript/ESPript).

A
U1

U1

Mg2+

Mg2+

L899
(C-term)3

L899(C-term)

2

3

Q645

2

Q645

B

Y603

Y603

3.4 Å

3.3 Å

Y603

g1C

g1U

Y603

g1A

g1G

D

C

3

3

1

4

5

U1

2

2

1

L2

5

4

3

2

MID
MID
D

4

5

PIWI
PIW
W

Figure S6. Recognition of the Guide 50 Segment, Related to Figure 3(A) mFO  DFC omit electron density map for the bound Mg2+ ion (green mesh, contoured at 10s).
(B) g1U recognition by Siwi. The specificity loop in the MID domain is highlighted in orange. The modeled A, G and C at the g1 position would form steric clashes orrepulsion with Tyr603 in the specificity loop. The favorable and unfavorable interactions are indicated by cyan and red dashed lines, respectively.
(C) Superimposition of the 50 segment of the Siwi-bound piRNA (red) onto an A-form RNA duplex (gray) (stereoview).
(D) Binding of the piRNA seed region to the Siwi MID-PIWI lobe.

A

B

Siwi PAZ

Miwi PAZ

Y417 Y379

F343
M382

F370

F333

Um28
Um8

C

D

Hiwi1 PAZ

hAgo2 PAZ

M381 F342

V308
Y338

F332

Um14

F294

U21

Figure S7. Recognition of the Guide 30 Segment, Related to Figure 4(A–D) PAZ domains of Siwi (A), Miwi (PDB: 2XFM) (B), Hiwi1 (PDB: 3O7V) (C), and hAgo2 (PDB: 4W5N) (D). The 20 -O-methyl group is indicated by red arrows.