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

Abstract

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. 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. RNA interference, an ancient mechanism of gene silencing mediated by small RNAs, is widely distributed throughout most eukaryotes (1Meister G. Tuschl T. Nature. 2004; 431: 343-349Crossref PubMed Scopus (1961) Google Scholar, 2Hannon G.J. Nature. 2002; 418: 244-251Crossref PubMed Scopus (3497) Google Scholar). Potent silencing of genes is achieved through the processing of double-stranded RNA (dsRNA) 2The abbreviations used are: dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; RISC, RNA-induced silencing complex; AaAgo, Aquifex aeolicus Argonaute; ssDNA, single-stranded DNA. 2The abbreviations used are: dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; RISC, RNA-induced silencing complex; AaAgo, Aquifex aeolicus Argonaute; ssDNA, single-stranded DNA. into small interfering RNAs (siRNA) by the enzyme Dicer. The ∼22-nt-long siRNAs contain a signature 5′-phosphate group and a 2-nt-long 3′-overhang (3Bernstein E. Caudy A.A. Hammond S.M. Hannon G.J. Nature. 2001; 409: 363-366Crossref PubMed Scopus (3745) Google Scholar). The siRNA is then loaded into the RNA-induced silencing complex (RISC), of which Argonaute is the primary catalytic component (4Liu J. Carmell M.A. Rivas F.V. Marsden C.G. Thomson J.M. Song J.J. Hammond S.M. Joshua-Tor L. Hannon G.J. Science. 2004; 305: 1437-1441Crossref PubMed Scopus (2018) Google Scholar, 5Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1435) Google Scholar). Energetic asymmetry of the siRNA ends allows for its directional loading into RISC (6Khovorova A. Reynolds A. Jayasena S.D. Cell. 2003; 115: 209-216Abstract Full Text Full Text PDF PubMed Scopus (1995) Google Scholar, 7Schwarz D.S. Hutvanger G. Du T. Xu Z. Aronin N. Zamore P.D. Cell. 2003; 115: 199-208Abstract Full Text Full Text PDF PubMed Scopus (2176) Google Scholar, 8Tomari Y. Matranga C. Haley B. Martinez N. Zamore P.D. Science. 2004; 306: 1377-1380Crossref PubMed Scopus (459) Google Scholar). Argonaute cleaves the passenger strand of the siRNA, leaving the guide strand of the siRNA bound to RISC (9Rand T.A. Petersen S. Du F. Wang X. Cell. 2005; 123: 621-629Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 10Matranga C. Tomari Y. Shin C. Bartel D.P. Zamore P.D. Cell. 2005; 123: 607-620Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 11Gregory R.I. Chendrimada T.P. Cooch N. Shiekhattar R. Cell. 2005; 123: 631-640Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar). This single-stranded guide strand siRNA bound to Argonaute is required for the sequence-dependent recognition of mRNA by RISC. Recombinantly expressed human Argonaute 2 in complex with single-stranded siRNA forms a minimal RISC, capable of mRNA recognition and cleavage (12Rivas F.V. Tolia N.H. Song J.J. Aragon J.P. Liu J. Hannon G.J. Joshua-Tor L. Nat. Struct. Mol. Biol. 2005; 12: 340-349Crossref PubMed Scopus (548) Google Scholar). Recent crystal structures of Argonaute apoenzymes show the architecture of Argonaute to be a multidomain protein composed of N-terminal, PAZ, MID, and PIWI domains (13Song J.J. Smith S.K. Hannon G.J. Joshua-Tor L. Science. 2004; 305: 1434-1437Crossref PubMed Scopus (1081) Google Scholar, 14Yuan Y.R. Pei Y. Ma J.B. Kuryavyi V. Zhadina M. Meister G. Chen H.Y. Dauter Z. Tuschl T. Patel D.J. Mol. Cell. 2005; 19: 405-419Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The catalytic cycle of RISC requires the binding of a number of different nucleotide structures to Argonaute, and we expect Argonaute to undergo a number of conformational changes during the cycle of mRNA recognition by RISC (15Filipowicz W. Cell. 2005; 122: 17-20Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 16Tomari Y. Zamore P.D. Genes Dev. 2005; 19: 517-529Crossref PubMed Scopus (735) Google Scholar). Nevertheless, it remains unclear how the multidomain arrangement of Argonaute recognizes and distinguishes between single-stranded and double-stranded oligonucleotides that correspond to the Dicer-processed siRNA product, guide strand siRNA, and the guide strand/mRNA duplex. We report the crystal structure of Aquifex aeolicus Argonaute (AaAgo), crystallized in the presence of a single-stranded siRNA and refined using data to 3.2 Å resolution. We propose a model for Argonaute activity in the context of the RISC complex. We also report a kinetic and 5.5-ns molecular dynamics characterization of Argonaute, which highlights biphasic binding kinetics with ssDNA, and a tendency for pronounced motion of the PAZ and N-terminal domains. Together, these studies highlight a functional role for domain flexibility, especially of the PAZ, in Argonaute activity that may be crucial for the function of the RISC. Protein Expression and Purification—The full-length Argonaute gene from Aquifex aeolicus was amplified by PCR from genomic DNA and cloned into a modified pET16b vector (Novagen) containing an N-terminal His7 tag. AaAgo was overexpressed in Escherichia coli host cell strain BL21 (DE3) pLysS (Stratagene) and induced by 1 mm isopropyl-1-thio-β-d-galactopyranoside (A600 ∼ 0.8) at 37 °C for 3 h. Cells were resuspended and sonicated in 10 mm Hepes, 300 mm NaCl, pH 7.4. Non-thermostable proteins were removed by a heating step of 72 °C for 30 min. Precipitated proteins were removed by centrifugation at 20,000 rpm, and the supernatant was applied to an immobilized metal affinity column (nickel-nitrilotriacetic acid-agarose; Qiagen), with the partially purified target protein eluting at 300 mm imidazole. The N-terminal His7 tag was removed by addition of TEV protease overnight at 4 °C. The target was further purified by gel filtration on a Superdex 200 column (Amersham Biosciences), equilibrated with 10 mm Hepes, 300 mm NaCl, pH 7.4. For binding experiments, the protein was purified by affinity chromatography and gel filtration in 20 mm Hepes, 150 mm KCl, pH 7.4. Crystallization, Data Collection, and Refinement—siRNA nucleotides (5′-PO4 GGG AAU GCG ACU CAU GAA GCU-3′) were purchased from Ambion and solubilized in 10 mm Hepes, pH 7.4. An equimolar ratio of protein at 10 mg/ml and single-stranded siRNA was mixed together and incubated at 37 °C for 15 min prior to crystallization trials. Crystals were grown by the hanging drop method at 22 °C, with 3.0 m sodium formate, 100 mm Hepes, pH 7.4, as the precipitant. Crystals grew within 2 weeks to a maximum size of ∼200 × 200 × 30 μm. For cryoprotection, crystals were transferred into mother liquor containing 25% glycerol (v/v) and immediately frozen in liquid nitrogen. Data were collected at beamline ID23-1 at European Synchrotron Radiation Facility in 1° oscillations. Diffraction was observed to a useable resolution of 3.2 Å and indexed in tetragonal space group I422, with one molecule/asymmetric unit. Data were processed using MOSFLM and SCALA from the CCP4 suite (17Bailey S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar). Although the data were weak, with reflections at an I/σ(I) of 2 at 3.6 Å resolution, a plot of the SigmaA versus resolution (supplemental Fig. S1) showed that the weak data were still usable, and thus the refinement utilized all data to 3.2 Å resolution (18De La Barre B. Brunger A.T. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 932-1032Google Scholar, 19Ling H. Boodhoo A. Hazes B. Cummings M.D. Armstrong G.D. Brunton J.L. Read R.J. Biochemistry. 1998; 37: 1777-1788Crossref PubMed Scopus (370) Google Scholar). The structure was solved by molecular replacement, using 1YVU as a search model. Although an unambiguous solution was initially obtained by the CCP4 program MOLREP (17Bailey S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar), the density for the PAZ domain was poor. A second run using 1YVU excluding the PAZ domain was done in MOLREP, while placement of the PAZ domain was facilitated by EPMR (20Kissinger C.R. Ghlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (690) Google Scholar). The model was refined by a combination of rigid body, positional, simulated annealing, and grouped and individual B-factor refinement in A.T. P.D. J. M. Read R.J. T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; PubMed Scopus Google with in Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). B-factor it a number of is in and is by the of the at resolution (18De La Barre B. Brunger A.T. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 932-1032Google Scholar). of grouped and individual B-factor refinement individual B-factor refinement in the and grouped B-factor refinement in the Data and refinement in The of refinement especially in to B-factor is in in the and refinement for an of 2 at 3.6 Å cell in to in the resolution in to in the resolution in to in the resolution in to in the resolution in to in the resolution in to in the resolution in to in the resolution PIWI The for an of 2 at 3.6 Å in to in the resolution in a model was used for molecular dynamics of in the model were using M.A. A. R. F. A. Struct. PubMed Scopus Google Scholar). A 5.5-ns molecular dynamics simulation was then using the T. H. R. A. C. Wang B. R.J. J. 2005; PubMed Scopus Google suite of together with the and a for C. B. J. 2002; PubMed Scopus Google Scholar). The structure was into an of J. J. J. Scopus Google that the between the of the and the protein was at 10 The method T. L. J. Scopus Google was used to with a space of to were using J.P. G. J. Scopus Google Scholar). The step for the simulation was 2 the system was to 300 a of which the density was during applied during these to all were then an of simulation at 300 the were used for obtained 1 changes during the simulation were in of of from the structure and in the between at the of the of the of between and obtained T. M. PubMed Scopus Google Scholar). For and for a component of in space was that the dynamics of the system A. PubMed Scopus Google Scholar). The most in the observed conformational between 1YVU and the structure were then obtained by the between and to PubMed Scopus Google An of one that the of of all in the N-terminal PAZ domain were used for to the of J. Mol. Biol. PubMed Scopus Google Scholar, M. T. J. Mol. Biol. PubMed Scopus Google Scholar). Binding of DNA to immobilized AaAgo was in by total internal reflection fluorescence This was used in combination with interference for of the of immobilized AaAgo M. S. J. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of the protein the through its tag was achieved by using to a which were loaded with S. J. 2005; PubMed Scopus Google Scholar). 100 of and and with were used for all binding AaAgo in 20 mm Hepes, 150 mm KCl, pH 7.4, was immobilized the at between 1 and mm was in the The was with an at at were at °C with a DNA of by a protein was the by addition of mm imidazole. were using of is a molecule × × Å in all of the in the with the of and The domains of AaAgo into the N-terminal, PAZ, and the domains. The N-terminal domain is a mixed that of as by a search for A as at the between the a to by the N-terminal and PIWI domains and the N-terminal and PAZ domains. The PAZ domain an architecture to that of PAZ domain structures, into and The PAZ domain recognizes the 3′-overhang of the guide strand and structures of show the nucleotide between and of PAZ A. B. E. M. Nat. Struct. Biol. 2004; PubMed Scopus Google Scholar, J.B. Y.R. Meister G. Pei Y. Tuschl T. Patel D.J. Nature. 2005; PubMed Scopus Google Scholar). Although PAZ domains the binding cleft for the of the is and is mediated by residues. AaAgo, these and within the of PAZ. A is in recognition and of the guide strand A second the PAZ domain to the by the and PIWI domains and The binding pocket for the 5′-phosphate of the guide strand at the between the and PIWI domains. This binding pocket is Argonaute and PIWI and by the and and the of the protein by the PIWI pocket in The PIWI domain an to as and L. H. R. Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. A. PubMed Scopus Google and as in structures of Argonaute and PIWI of the target mRNA strand through a mechanism as mediated by as (12Rivas F.V. Tolia N.H. Song J.J. Aragon J.P. Liu J. Hannon G.J. Joshua-Tor L. Nat. Struct. Mol. Biol. 2005; 12: 340-349Crossref PubMed Scopus (548) Google Scholar). RNA Binding architecture of structure in a Y-shaped system of grooves that throughout the protein The of the is by the PAZ and domains and the catalytic of the of the and the the the composed of the guide strand siRNA and the target (13Song J.J. Smith S.K. Hannon G.J. Joshua-Tor L. Science. 2004; 305: 1434-1437Crossref PubMed Scopus (1081) Google Scholar). structures of PIWI bound to model J.B. Y.R. Meister G. Pei Y. Tuschl T. Patel D.J. Nature. 2005; PubMed Scopus Google Scholar, S.M. Nature. 2005; PubMed Scopus Google Scholar). our the Å in the of and within Å of one thus a conformational rearrangement to the The PAZ in an that of the a the of a crystal structure of AaAgo was Y.R. Pei Y. Ma J.B. Kuryavyi V. Zhadina M. Meister G. Chen H.Y. Dauter Z. Tuschl T. Patel D.J. Mol. Cell. 2005; 19: 405-419Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), crystallized in a different space group from to as The between the structures most pronounced in the of the PAZ This from a of the the N-terminal and PAZ domains. of the conformational changes between our structure and 1YVU using S. 1998; PubMed Scopus Google shows a in the of the PAZ in our from the N-terminal domain domain in our structure the binding for the of the guide strand The of the PAZ domain observed in our structure the of the Argonaute which also a structure with grooves between the N-terminal domain and the PAZ and PIWI domains (13Song J.J. Smith S.K. Hannon G.J. Joshua-Tor L. Science. 2004; 305: 1434-1437Crossref PubMed Scopus (1081) Google Scholar). The of the PAZ with for the of the further a tendency of the domain to be the in the PAZ domain with an of Å we a to how the structure of the PAZ domain is by the density The was by a of the model in the and the to simulated The shows density for the PAZ domain (supplemental Fig. that the of the PAZ domain is in our We also that in the crystal the PAZ domain is by crystal which may be the of the the PAZ domain of 1YVU is in crystal with the N-terminal, MID, and PIWI domains. the conformational in the AaAgo structures the to the nucleotide binding in PAZ, a functional of the PAZ domain in RISC An of the of the AaAgo structures shows that and in the of the N-terminal domain in our structure to 1YVU and in our structure versus and in and the to the nucleotide binding pocket in PAZ, also in our structure and in our structure versus and in of the Y-shaped is by the N-terminal and PIWI domains and Å in The size of the is in 1YVU as as The is with basic which may to the of The of to the PIWI a in Argonaute at one of the Although the of the PIWI in human the is and is at the of the by the PIWI and N-terminal domains. A between and Argonaute was to an between the PIWI and the second domain of N. H. W. 2004; PubMed Scopus Google Scholar, F. A. P.D. Science. 2006; PubMed Scopus Google Scholar), while studies that Argonaute is for the cleavage of the strand of Dicer-processed siRNA (9Rand T.A. Petersen S. Du F. Wang X. Cell. 2005; 123: 621-629Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 10Matranga C. Tomari Y. Shin C. Bartel D.P. Zamore P.D. Cell. 2005; 123: 607-620Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar). these the of within the RISC complex and that the may as a channel for of the siRNA into the of The second of the Y-shaped is by the N-terminal and PAZ domains and Å in The between the N-terminal and PAZ domains in our structure between the of and which at the of the in our the binding pocket in PAZ is to the 1YVU shows a by a between the of and as as a between the of and the of structure shows between these with a of Å and a of the size of the shows between our structure and the size of the is the A of the between AaAgo and human and a number of from the N-terminal and and from the PIWI which the of the basic in the and as a RNA recognition of the N-terminal the and grooves Although AaAgo was crystallized in the presence of a single-stranded siRNA, density was observed in the binding with density observed in the binding in PAZ. This may be by the of AaAgo for ∼ to ∼ as by binding Y.R. Pei Y. Ma J.B. Kuryavyi V. Zhadina M. Meister G. Chen H.Y. Dauter Z. Tuschl T. Patel D.J. Mol. Cell. 2005; 19: 405-419Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). further the of the PAZ domain within Argonaute, we a 5.5-ns molecular dynamics of the a tendency for the PAZ and to a the N-terminal to to a and of the This is by a of of from the structure the domains show that the N-terminal and PAZ domains show conformational changes of to and was a tendency for the PAZ and N-terminal domains to and during the of the with and a observed for the PAZ and at for the N-terminal domain together with the conformational show an of these a of the structure during the The to changes of the between domains, as in Fig. for of at the that show in our structure 1YVU in the of to 10 Å be which to changes in the of the of between of in Fig. The shows within the PAZ and N-terminal domains, indicative of a rigid of motion of the domains. a component of the of the was for the of the molecular dynamics A. PubMed Scopus Google Scholar). show that protein motion is by a small number of S. N. PubMed Scopus Google Scholar). for is that protein structures that the the of Struct. Biol. 2005; 15: PubMed Scopus Google Scholar). number 2 and number 4 showed the in the observed conformational between 1YVU and the structure in with to of the observed motion together with the of is as it that the observed for the structure may to conformational that of the 1YVU a of Argonaute may in the of the of Binding to further the between and Argonaute, we kinetic binding using immobilized We used fluorescence by total internal reflection fluorescence The is by the from the The of containing a 5′-phosphate with immobilized AaAgo shows biphasic and kinetics that the was to binding to the a with 100 in the of immobilized protein was minimal fluorescence The kinetics was of the immobilized protein and to and 10 protein the The be with a with a of to of the from Argonaute of the on the of AaAgo was which the that of the with immobilized protein was the of the binding a protein and the is the ratio of the and The kinetic observed in the of AaAgo and a for the Nevertheless, a of be on the of the of the and The was to be between and in with the by Y.R. Pei Y. Ma J.B. Kuryavyi V. Zhadina M. Meister G. Chen H.Y. Dauter Z. Tuschl T. Patel D.J. Mol. Cell. 2005; 19: 405-419Abstract Full Text Full Text PDF PubMed Scopus (293) Google by binding We that the nucleotide and were used for these kinetic with immobilized We then the kinetics of binding as a function of the of ssDNA, using and in addition to the in the the kinetic is of the of used of is that the of binding is to the of the in that a of PAZ is by our crystal structure and our molecular dynamics simulation that the of Argonaute a number of a and and The molecular dynamics simulation thus further to the of a PAZ domain to a the N-terminal is from the different of AaAgo in our structure and as as the structure of the and forms of Argonaute may that in upon nucleotide binding. The biphasic kinetics of binding to AaAgo is further of a conformational rearrangement in the We that the fluorescence observed is to the of the with the protein and to the The total internal reflection fluorescence spectroscopy show in the and kinetics of and nucleotides We the from that the of the is to the at the and that the DNA may be with the kinetics of the also of the of used and the of immobilized with the data from AaAgo crystal structures and the molecular dynamics the kinetic domain during binding and as by the changes in DNA fluorescence data with the crystal structures of the which shows nucleotide density for the J.B. Y.R. Meister G. Pei Y. Tuschl T. Patel D.J. Nature. 2005; PubMed Scopus Google Scholar, S.M. Nature. 2005; PubMed Scopus Google Scholar). of PAZ and data in with the 1YVU and Argonaute structures, an to at conformational changes in Argonaute during the cycle of mRNA recognition and cleavage by RISC. the structures of Argonaute from different with the structure allows for the the of Argonaute structures from the the context of the RISC, which as a complex composed of Argonaute, and R.I. Chendrimada T.P. Cooch N. Shiekhattar R. Cell. 2005; 123: 631-640Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar), our structure and data on the and for the loaded Dicer-processed siRNA as as the is that the loading of the Dicer-processed siRNA may through the prior to Argonaute cleavage of the passenger The of the PIWI to the and the between the PIWI and the catalytic domains of The of the may therefore to the recognition of the siRNA in the complex. cleavage of the passenger strand in the loading the single-stranded guide complex is in the We that Argonaute is most in the PAZ of the the of the PAZ domain may be This may for recognition of the target mRNA as as between and in the The mRNA strand may be to the and the enzyme through the between the PAZ and N-terminal domains the between the N-terminal and PIWI domains. This to be by further and The structure together with our molecular dynamics and kinetic a critical role for PAZ in the function of and studies be to further the cycle of siRNA loading and mRNA recognition and cleavage by We the at European Synchrotron Radiation Facility beamline ID23-1 for with data and and for critical on the We and for the with