Two symmetric arginine residues play distinct rolesin Thermus thermophilus Argonaute DNA guidestrand-mediated DNA target cleavageJinping Leia, Gang Shengb, Peter Pak-Hang Cheunga, Shenglong Wangc, Yu Lid, Xin Gaod, Yingkai Zhangc,e,1,Yanli Wangb,1, and Xuhui Huanga,f,1

aDepartment of Chemistry, Center of Systems Biology and Human Health, State Key Laboratory of Molecular Neuroscience, Hong Kong Branch of ChineseNational Engineering Research Center for Tissue Restoration & Reconstruction, The Hong Kong University of Science and Technology, 190, Clear Water Bay,Kowloon, Hong Kong; bKey Laboratory of RNA Biology, Chinese Academy of Sciences Center for Excellence in Biomacromolecules, Institute of Biophysics,Chinese Academy of Sciences, 100101 Beijing, China; cDepartment of Chemistry, New York University, New York, NY 10003; dComputational BioscienceResearch Center, Computer, Electrical and Mathematical Sciences and Engineering Division, King Abdullah University of Science and Technology,23955-6900 Thuwal, Saudi Arabia; eNew York University–East China Normal University Center for Computational Chemistry, New York University Shanghai,200062 Shanghai, China; and fHong Kong University of Science and Technology–Shenzhen Research Institute, 518057 Shenzhen, China

Edited by Donald G. Truhlar, University of Minnesota, Minneapolis, MN, and approved November 29, 2018 (received for review October 4, 2018)

Bacterium Thermus thermophilus Argonaute (Ago; TtAgo) is a pro-karyotic Ago (pAgo) that acts as the host defense against theuptake and propagation of foreign DNA by catalyzing the DNAcleavage reaction. The TtAgo active site consists of a plugged-inglutamate finger with two arginine residues (R545 and R486)located symmetrically around it. An interesting challenge is tounderstand how they can collaboratively facilitate enzymaticcatalysis. In Kluyveromyces polysporus Ago, a eukaryotic Ago,the evolutionarily symmetrical residues are arginine and histidine,both of which function to stabilize the plugged-in catalytic tetradconformation. Surprisingly, our simulation results indicated that,in TtAgo, only R545 is involved in the cleavage reaction by servingas a critical structural anchor to stabilize the catalytic tetrad Asp-Glu-Asp-Asp that is completed by the insertion of the glutamatefinger, whereas R486 is not involved in target cleavage. The TtAgo-mediated target DNA cleavage occurs in a substrate-assisted mech-anism, in which the pro-Rp (Rp, a tetrahedral phosphorus centerwith “R-type” chirality) oxygen of scissile phosphate acts as a gen-eral base to activate the nucleophilic water. Our unexpected theo-retical findings on distinct roles played by R545 and R486 in TtAgocatalysis have been validated by single-point site-mutagenesis ex-periments, wherein the target cleavage is abolished for all mutantsof R545. In sharp contrast, the cleavage activity is maintained for allmutants of R486. Our work provides mechanistic insights on thecatalytic specificity of Ago proteins and could facilitate the designof new gene-editing tools in the long term.

bacterial Argonaute | QM/MM simulations | DNA cleavage

Argonaute (Ago) proteins are critical components of theRNA-induced silencing complex that play an essential rolein guide strand-mediated target RNA recognition and cleavage(1–15). The prokaryotic Ago (pAgo) proteins are characterizedto serve as host defense against the uptake and propagation offoreign RNA/DNA through RNA/DNA interference (16–20),whereas eukaryotic Ago (eAgo) proteins carry out this process ofhost defense through RNA interference (20, 21). Molecular in-sights into target DNA/RNA cleavage have emerged from struc-tural (13) and chemical studies (16, 22), with potential applicationas gene manipulation against a range of diseases (9, 21, 23–32).Bacterium Thermus thermophilus Ago (TtAgo), as reported in

our previous structural work (13), features four domains (PIWI,MID, PAZ, N) and two connecting linkers (L1, L2) as found inother Ago proteins (13, 16, 19, 21). The PIWI domain adopts anRNase H fold, in which the catalytic Asp-Glu-Asp-Asp tetradcontributes to the slicer activity (13, 21, 33–37). Previous phos-phorothioate substitution cleavage kinetic studies (5, 13) alsosuggested that TtAgo-mediated target DNA cleavage might followthe RNase H-mediated two-metal-ion catalysis cleavage pathway

(38–46). However, distinct from RNase H, whose catalytic tetradis formed during initial folding, Ago proteins require insertion ofthe glutamic acid residue on loop PL2 (termed “glutamate fin-ger”) into the catalytic pocket when it is bound to the guide/targetnucleotide strand (13, 21, 37). This difference is likely a result ofthe high catalytic specificity of Ago; to the contrary, RNase Hexerts a nonspecific role in RNA–DNA hybrid cleavage (37).Also, the TtAgo-mediated target DNA cleavage is a typicalphosphoryl transfer reaction (45, 47–52) that requires a generalacid to protonate the leaving group and a general base todeprotonate the inline nucleophilic-attacking water under neu-tral pH (13, 16, 40, 44). Importantly, it is unclear whether thisplugged-in glutamate finger could act as a general acid to directlyparticipate in cleavage reaction or only act as a structural an-chor to impart the catalytic specificity of Ago. In addition, it isunknown how the two-metal-ion catalysis and pro-Rp (Rp, atetrahedral phosphorus center with “R-type” chirality) oxygenatoms achieve substrate specificity of TtAgo (45).

Significance

Argonaute (Ago) proteins function in host defense against for-eign mRNA. The Ago-mediated mRNA target cleavage necessi-tates glutamate finger insertion. Two positively chargedresidues locate symmetrically around the plugged-in finger, buttheir functional roles are unclear for Thermus thermophilus Ago(TtAgo), a prokaryotic Ago protein. Surprisingly, our simulationsand site-mutagenesis experiments indicated that, in contrast tothe equivalent roles of symmetrical positively charged residuesin Kluyveromyces polysporus Ago (a eukaryotic Ago protein), inTtAgo, only R545 is critical for mRNA cleavage as a structuralanchor to stabilize the plugged-in conformation, whereasR486 plays a negligible role. These differences provide a mo-lecular basis of the distinct glutamate finger functions of Agoproteins in different organisms.

Author contributions: Y.Z., Y.W., and X.H. designed research; J.L., G.S., Y.L., and X.G.performed research; S.W. and Y.Z. contributed new reagents/analytic tools; J.L., G.S.,P.P.-H.C., Y.W., and X.H. analyzed data; and J.L., G.S., P.P.-H.C., Y.Z., Y.W., and X.H. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: yz22@nyu.edu, ylwang@ibp.ac.cn, orxuhuihuang@ust.hk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1817041116/-/DCSupplemental.

Published online December 27, 2018.

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The Ago-mediated cleavage generally necessitates glutamatefinger insertion, and this plugged-in conformation can be stabi-lized through a hydrogen-bond network in pAgo and eAgoproteins as observed in the reported crystal structures (13, 21, 37,53–55). Interestingly, the two symmetric positively charged res-idues that form hydrogen bonds with plugged-in glutamate fingerare arginine in pAgo (13), such as R545 and R486 in TtAgo (13),whereas, in eAgo (37, 53, 54), they are arginine and histidine,both of which function to stabilize the plugged-in conformationof Kluyveromyces polysporus Ago (KpAgo) (37). It is unknownfrom the previously reported ternary structure (13) whether thetwo symmetrical arginines in TtAgo play the same roles as thecorresponding arginine and histidine in KpAgo (37). Further-more, an intriguing question is whether this structural differencehas mechanistic consequences that explain the evolutionary dif-ferences between prokaryotic and eAgo proteins.Interestingly, in the present work, we found that, in contrast to

the equivalent roles of symmetrical Arg and His residues inKpAgo (37), the two seemingly symmetric residues in TtAgo,R545 and R486, play distinct roles during target cleavage.Specifically, R545 is a key structural anchor in stabilizing thecatalytic plugged-in conformation for target cleavage, whereasR486 is not involved in cleavage reaction. Through extensive abinitio quantum mechanics (QM)/molecular mechanics (MM)molecular dynamics (MD; aiQM/MM-MD) simulations withperiodic boundary conditions (56), we characterized a substrate-assisted (57, 58) reaction pathway for this TtAgo-mediated targetDNA cleavage (the system contains more than 170,000 atoms inMM region and 67 atoms in QM region; Fig. 1A). Our resultsrevealed that the plugged-in glutamate finger (E512) does notact as a general acid to protonate the 3′ leaving group, but onlyserves as a critical structural anchor to impart the catalyticspecificity of TtAgo. Our theoretical predictions on the roles ofcritical residues have been further validated by in vitro cleavageassays performed on R545 and R486 single-point mutants.

Results and DiscussionIn this work, we carried out B3LYP(6-31G*) QM/MM-MD (56)simulations coupled with experimental measurements of cleav-age activity for selective mutants to characterize the detailedreaction mechanism for the TtAgo-mediated target DNAcleavage and identify the catalytic roles of some critical residues,such as E512, R545, R486, and K575. Given that R486 andR545 are located symmetrically around the plugged-in glutamatefinger E512 in the precleavage state (13), we discovered that theyplayed distinct catalytic roles during target cleavage.

TtAgo-Mediated DNA Target Cleavage Follows a Substrate-AssistedCleavage Pathway.Our aiQM/MM-MD simulation results revealedthat the TtAgo-mediated target DNA cleavage exhibits a substrate-assisted (57, 58) mechanism (Fig. 1B and C), in which the scissilephosphate acts as the general base to deprotonate the nucleophilicwater and the protonation of 3′ leaving oxygen is accomplished byshuttling the attacking water proton through the pro-Rp oxygen ofscissile phosphate, similar to the two-metal-ion catalysis used byRNase H (45, 59) and ribozyme (46, 60). Our calculations indicatedpentacovalent phosphorus as intermediates experimentally (61–63) and theoretically (38, 44). Also, the free energy profiles (Fig. 1 CandD and SI Appendix, Fig.S1) computed from umbrella sampling(64, 65) found that the rate-determining step is the nucleophilicattack of water to form the pentacovalent phosphorus intermediate,with a free energy barrier of 16.8 ± 0.2 kcal/mol (kcat = 112.3 ±0.8 s−1), similar to that of RNase H-mediated RNA cleavage (38,44, 66, 67). In addition, our pH-dependent experimental results(Fig. 1E) indicated that the TtAgo-mediated target DNA cleavageoccurs at neutral pH condition, which strongly supports ourcalculated substrate-assisted mechanism that does not requireany amino acid residues of the enzyme to have specific protonation

state to support catalytic activity. Our proposed detailed cleavagemechanism explains how substrate specificity for the Ago-mediatedDNA/RNA cleavage is achieved.We decipher from the previous structure (13) a “substrate-as-

base” mechanism (42, 45, 46, 57, 58, 68, 69) for the target DNAcleavage reaction, and our simulation results showed that it is thepro-Rp oxygen of scissile phosphate, rather than the pro-Rpoxygen of phosphate group 3′ to scissile bond, that acts as ageneral base for the target cleavage. This is because it is notenergetically feasible for the pro-Rp oxygen of phosphate group3′ to scissile bond to deprotonate the nucleophilic water (SIAppendix, Fig. S2). Instead, the role of pro-Rp oxygen of phos-phate group 3′ to scissile bond is to orient and stabilize theattacking hydroxide ion through hydrogen bond networks (Fig.1B). In addition, when the pro-Rp oxygen of scissile phosphatewas substituted to sulfur, the energy barrier for the target DNAcleavage reaction was much higher (SI Appendix, Fig. S3). Thesecalculation results are in accordance with the previous reportedexperimental observations that the cleavage rate was reduced by200 fold for phosphorothioate substitution of pro-Rp oxygen atthe scissile phosphate, whereas the rate was reduced by only15 fold for phosphorothioate substitution of pro-Rp oxygen atthe phosphate group 3′ to scissile bond (5).It is obvious from our simulation results that the TtAgo-

mediated target DNA cleavage has no apparent general acidto donate the proton for the 3′ leaving group of cleavage atneutral pH (13, 45) (SI Appendix, Figs. S4 and S5). Neither of thetwo remaining candidates for general acid, the plugged-in glu-tamate finger E512 and the positive charged K575, could pro-tonate the 3′ leaving group. It is because the carboxylic hydrogenof protonated E512 does not form a hydrogen bond with bridgingwaters (SI Appendix, Fig. S4B), and the energy barrier is too highfor the proton transfer from protonated E512 to the 3′ leavingoxygen through bridging water (SI Appendix, Fig. S4C). Addi-tionally, K575, a better candidate than E512 because it locatescloser to 3′ leaving oxygen, cannot act as a general acid becauseit is not energetically feasible for the proton transfer fromK575 to the 3′ leaving oxygen (SI Appendix, Fig. S5).

The Plugged-In Glutamate Finger E512 only Acts as a CriticalStructural Anchor to Form Catalytic Tetrad Conformation. As dis-cussed here earlier, the plugged-in glutamate finger does not actas a general acid. Instead, the highly conserved glutamate fingerE512 is a crucial structural anchor for the formation of catalytictetrad from the previously reported structures (Fig. 2A and SIAppendix, Fig. S6) (13), and our aiQM/MM-MD simulation re-sults indicated that the plugged-in catalytic tetrad conformationis well maintained throughout the whole target cleavage process(Fig. 1B and SI Appendix, Table S1). To further validate the roleof glutamate finger in structural anchoring, we performed clas-sical MD simulations and DNA-cleavage experiments for mu-tants E512A and E512Q. The simulation results show that thecatalytic active site configuration is disrupted for mutants E512Aand E512Q in the precleavage state, as the distances betweenMg2+ A and B (Fig. 2 B and D and SI Appendix, Table S2) arelarger than 4.5 Å, which exceeds the separation distance (

Fig. 1. The substrate-assisted target DNA cleavage mechanism. (A) Overview of the setup used for our simulations for TtAgo-mediated target DNA cleavage.Snapshot of the simulation box (Left) and detailed view of the QM subsystem in the precleavage state (Right; PDB ID code 4NCB). (B) Detailed cleavagemechanism (Top) and respective critical structures (Bottom). Movies S1 and S2 show the mechanism details. The coordinating amino acid residues D546, D478,D660, and E512 are colored dark gray, and the coordinating waters that do not participate in the cleavage reaction are colored light gray. INT1, first in-termediate; INT2, second intermediate; PC, product complex, i.e., postcleavage state; RC, reactant complex, i.e., precleavage state; TS1, first transition state;TS2, second transition state; TS3, third transition state. (C) Calculated free energy changes with their statistical errors along the reaction progress (in kilo-calories per mole). Approximately 430 windows were selected to perform a total of approximately 6.5 ns B3LYP(6-31G*) QM/MM-MD simulations to generatethe free energy profile. (D) Calculated 2D PMF along the reaction progress (in kilocalories per mole). The reaction coordinate d1 involves the nucleophilicattack of water to form a new P–O bond, reaction coordinate d3 − d4 is the hydrogen transfer from nucleophilic water to the pro-Rp phosphate oxygen oftarget nucleotide T10′, reaction coordinate d2 is the breaking of P–O bond between target nucleotides T10′ and C11′, and reaction coordinate d4 − d5 is theproton transfer from the pro-Rp oxygen of nucleotide T10′ to the 3′ leaving oxygen of target nucleotide C11′. (E) pH-dependent experimental measurementsfor the cleaved product of WT.

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D660) are also critical structural anchors, as the cleavage activ-ities are abolished for the mutants of these residues (SI Appen-dix, Fig. S9) (5, 6, 13). Hence, we conclude that the plugged-inglutamate finger is only a critical structural anchor to completethe catalytic tetrad to impart catalytic specificity for Ago-mediated target cleavage.

Two Symmetric Arginine Residues Play Distinct Roles: R545, Not R486,Is Involved in Target Cleavage as an Important Stabilizing StructuralSupport for the Catalytic Tetrad Conformation. Our simulation andexperimental results show that R545 is involved in target cleav-age as a crucial stabilizing residue for the plugged-in catalytictetrad conformation. In the precleavage state, for mutantsR545A, R545K, and R545Y, the simulated distances betweenMg2+ A and B (Fig. 3 B and D and SI Appendix, Table S2) aremuch larger than the separation distance (

remain almost unchanged in the precleavage state for all ofthe R486 mutants (including R486A, R486K, and R486Y mu-tants). For example, the distance between Mg2+ A and B andboth of their coordination geometries are mostly maintained.Furthermore, the hydrogen bonds between glutamate finger andbridge waters are also almost maintained in precleavage stateand INT1 state obtained from aiQM/MM-MD simulations.Consistently, our experimental results show that, in contrast toR545, there remained DNA cleavage activities for theseR486 mutants (Fig. 3C and SI Appendix, Fig. S8). Therefore, ourresults suggest that, drastically differently than R545, the seem-ingly symmetric R486 may not act as a critical structure anchor tomaintain plugged-in conformation.Interestingly, in contrast to RNase H, Ago-mediated cleavage

requires glutamate finger insertion to complete the catalytictetrad and impart catalytic specificity (21, 37). In pAgo, such asTtAgo (13, 21), the glutamate finger is inserted within the DDDtriad in the guide-target bound structure following guide-targetbase pairing (55), and both of the two symmetric arginine resi-dues may play roles in stabilizing the catalytic tetrad conforma-tion from the previously reported crystal structure (Fig. 3A) (13).However, from aiQM/MM-MD and MD simulations, we showthat, surprisingly, only R545 is involved in the cleavage reactionby serving as a critical structural anchor to stabilize the plugged-in conformation, whereas R486 is not directly involved incleavage reaction. Our experimental observations also prove thatsubstitution of R545 will completely eliminate the target cleav-age, whereas the cleavage activity is retained for R486 substitu-tions. In addition, when we mutated R486 to histidine, the cleavageactivity was also retained for this R486H mutant from our simu-lation results (SI Appendix, Fig. S7).We postulate that, in TtAgo, R486 may play the role of fa-

cilitating mRNA target binding and glutamate finger insertionbecause R486 flips close to the catalytic pocket together with theinsertion of the glutamate finger loop. As shown in the crystalstructure before finger insertion (SI Appendix, Fig. S10C) (13),R486 has already formed stable interactions with the glutamatefinger via the hydrogen bonds but locates far from the active site.During finger insertion, R486 moves along with the glutamatefinger loop (especially E512) close to the active site (SI Appendix,Fig. S10A). In sharp contrast, R545 always stays close to theactive site during the entire process. We thus speculate thatR486 may facilitate the insertion of the glutamate finger loop,which subsequently leads to the formation of the catalyticallyactive conformation. In contrast, in eAgo, such as KpAgo (37),the glutamate finger has already been inserted at the stage of thesiRNA (i.e., guide) bound complex formation before the recog-nition of the mRNA target. This sequential order of eventssuggests that the glutamate finger insertion is unlikely to becorrelated with the mRNA target recognition in eAgo. Even forthe siRNA-bound complex formation, the two symmetric posi-tive charge residues, R1045 and H977, are not likely to facilitatethe glutamate finger insertion because they are already locatedclose to the catalytic pocket before the glutamate finger insertionas shown in the apoenzymatic structure of the eAgo Neurosporacrassa QDE-2 (SI Appendix, Fig. S10D) (72). On the con-trary, R1045 and H977 (Fig. 3A and SI Appendix, Fig.S10B) havebeen shown to be critical for catalysis, as their mutants (R1045Aand H977A) lead to significant reduction of the cleavage activ-ities based on the fluorescence measurements (37). Therefore, itis striking that the functions of R545 and R486 in TtAgo aredifferent from those of R1045 and H977 in KpAgo; the differ-ence between R486 and H977 is especially pronounced. Wespeculate that differences in the functions of the arginine inTtAgo and histidine in KpAgo (37) may result in the distinctinsertion motions of glutamate finger between pAgo and eAgo,and therefore could shed light on the evolution of Ago proteins.Consistently, our sequence alignment results also show that R545

is highly conserved among different eAgo and eAgo proteins,whereas R486 is not conserved (SI Appendix, Fig. S6).

K575 Acts as a Structural Anchor to Maintain the Active SiteConformation. As discussed, K575 does not act as a generalacid for target DNA cleavage. Instead, we postulate that it actsas a structural anchor to maintain the catalytic active confor-mation through hydrogen bonds with D546 and phosphates oftarget nucleotide (Fig. 4A) (13). When K575 is mutated to ALA(Alanine), GLU (Glutamine), and ARG (Arginine) in the pre-cleavage state, simulation results (Fig. 4 C and D) showed thattheir active site configuration fluctuates much larger than that ofWT. In addition, when we mutate K575 in the INT1 state, thetransferred water hydrogen spontaneously flips close to the 3′-hydroxyl oxygen of nucleotide 11′ (Fig. 4E), disrupting the activesite configurations of these three mutants. Consistently, experi-ments showed that DNA cleavage is shut down for these threemutants (Fig. 4B and SI Appendix, Fig. S8). Based on these re-sults, we conclude that the role of K575 is to maintain the activesite configuration through its hydrogen bond network during thewhole cleavage process.

ConclusionsIntriguingly, we discovered the distinct roles of two seeminglysymmetric arginines, R486 and R545, for TtAgo-mediated targetcleavage. Previous research focused on the active site structuralfeature of Ago (1, 3, 5–7, 21, 73), and the correspondingR1045 and H977 in KpAgo were both reported to be involved incleavage (37) and proposed to stabilize the plugged-in catalytictetrad conformation. In the present work, our simulation andexperimental results revealed that, in TtAgo, it is R545, rather

Fig. 4. The simulation measuring active site stability and experimental re-sults of K575 mutants in comparison with that of WT. (A) Critical distance todescribe the stability of the active site of TtAgo. (B) Experimental mea-surements for the cleaved products at 120 min. Original figures are shown inSI Appendix, Fig. S8. (C) Average distance between Mg2+ cation A and B (da),(D) average distance between Mg2+ cation B and 3′-hydroxyl oxygen oftarget nucleotide C11′ (db), and their statistical errors are calculated in theprecleavage state (RC). (E) Average distance between phosphate hydroxylhydrogen and 3′-hydroxyl oxygen of target nucleotide C11′ (d5; see Fig. 1D fordefinition) and their fluctuations calculated in INT1 state. The statistical errorswere computed by bootstrapping 10 independent trajectories 10 times withreplacement. The precleavage crystal structure (PDB ID code 4NCB) complexedwith guide and target DNA is used for our simulation.

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than R486, that is involved in target cleavage and acts as a crucialstructural anchor in stabilizing the plugged-in catalytic tetradconformation, given that the precleavage state is inactive and thecleavage is abolished for R545A, R545K, and R545Y mutants. Incontrast, R486 is not involved in cleavage reaction because thecleavage activity of R486A, R486K, and R486Y mutants are allmaintained. By extensive B3LYP(6-31G*) QM/MM-MD simu-lations, we also showed that the target DNA cleavage is char-acterized by a substrate-assisted mechanism with a barrier heightof approximately 16.8 kcal/mol, and the plugged-in glutamatefinger only acts as a critical structural anchor for the catalytictetrad formation rather than a general acid to protonate the 3′leaving group. Our substrate-assisted mechanism and validationof catalytic role of the glutamate finger could impart the catalyticspecificity of Ago proteins. Overall, our finding of unique rolesfor the two positively charged residues in TtAgo in contrast tothe equivalent roles for the corresponding residues in KpAgomay provide a molecular basis for the differences in glutamatefinger insertion motion between pAgo and eAgo proteins. InpAgo, the glutamate finger plugged-in catalytic tetrad confor-mation is formed following guide-target base pairing, whereas, ineAgo, the plugged-in conformation is formed before guide-targetbase pairing. This information could illuminate the evolutionaryjourney from pAgo to eAgo proteins.

MethodsBased on our previous reported ternary crystal structures (13) of TtAgocomplex with guide and 19-mer target DNA, we performed classic MDsimulations and then obtained proper snapshots for the subsequent aiQM/MM simulations. We employed B3LYP/6–31G(d) QM/MM-MD (56, 74) simu-lations with umbrella sampling (65, 75, 76), a computational tour de force tostudy biochemical reactions. This state-of-the-art computational approachprovides a first-principles description of chemical bond formation/breakingand dynamics of the enzyme active site while properly incorporating theeffects of heterogeneous and fluctuating protein environment, and hasbeen demonstrated to be powerful in characterizing the reaction mecha-nism for a number of complex systems (64, 74, 77–87). In addition, we alsoperformed classic MD simulations for selective mutants of reactant andpentacovalent intermediate to identify the catalytic roles of key chargedresidues near the active site. More computational details and experimentalmeasurement methods are presented in the following subsections.

Structure Preparation for Simulation. The ternary crystal structure [ProteinData Bank (PDB) ID code (13) 4NCB] of TtAgo complex with guide and 19-mertarget DNA in cleavage-compatible states was the basis for our enzyme-substrate model. The missing residues and atoms were added by usingMODELER (88–90), and the PO2 group of DC5 in target DNA chain D wasdeleted because it was unsolved and far away from active site. The partialcharges for the 5′-phosphorylated terminal basis DT5 of guide DNA werefitted with HF/6–31G(d) calculations by using the restrained electrostaticpotential module (91) in the Amber package. The protonation states of theionizable residues were determined at pH 7 based on pKa calculations viaPROPKA (92–95) and H++ (96, 97) programs. If these two programs pro-duced inconsistent predictions, the local hydrogen bonding network wouldbe taken into account. As a result, the histidine residues HIS379, HIS500, andHIS621 were protonated as HIP in the following MD and QM/MM simula-tions for TtAgo complex with guide and 19-mer target DNA.

Classical MD Simulation. For the classical MD simulations of TtAgo complexwith guide and 19-mer target DNA, the starting model was subjected tominimization of the hydrogen atoms that were added by LEAP module ofthe Amber 12 simulation package (98) with 600 steps of steepest descentfollowed by an additional 600 steps of conjugate gradient minimization.Then, the whole system was solvated into explicit TIP3P water (99) moleculesby using a cubic box with a 15-Å buffer distance between the box wall andits nearest solute atom, and five Na+ ions were added to neutralize thecharge. As a result, the whole system contains ∼170,000 atoms. The sub-sequent energy minimizations and equilibration MD simulations followedthe same state-of-the-art protocol as in our previous studies (74, 78). First,the solvent and counter ions were minimized with 2,500 steps of steepestdescent followed by a 2,500-cycle conjugate gradient minimization byrestraining the protein, DNA, and Mg2+ atoms with a restraint force con-

stant of 50 kcal·mol−1·Å−2. While gradually reducing this restraint to25 kcal·mol−1·Å−2, the solvent and counter ions were first minimized(1,000 steps of steepest descent followed by 1,000-cycle conjugate gradient),then equilibrated with a 100-ps NVT [constant number (N), volume (V), andtemperature (T)] MD simulation (temperature, 10 K) followed by another100-ps NPT [constant number (N), pressure (P), and temperature (T)] (pres-sure, 1 atm) MD simulation. Next, the system was heated from 10 K to 340 Kwith a 200-ps NVT simulation and equilibrated with a 100-ps NPT simulation,during which the restraint force constant was reduced to 10 kcal·mol−1·Å−2.Then, two sequential 100-ps NPT equilibration simulations were performedwith looser restraint force constants of 1 kcal·mol−1·Å−2 and 0.1 kcal·mol−1·Å−2,respectively. Finally, a 50-ns production simulation was conducted at a tem-perature of 340 K with the Berendsen thermostat method (100) and aconstant pressure of 1 atm coupled with isotropic position scaling. Five in-dependent equilibration and production MD simulations were carried out intotal with different initial velocities. In all MD simulations, the SHAKE al-gorithm (101) for bond constraint and a time step of 1 fs were used, thelong-range electrostatic interactions were treated with particle mash Ewald(PME) (102, 103) method, and a 12-Å cutoff was used for van der Waals(vdW) and short-range PME interactions. All MD simulations were per-formed by using the Amber 12 MD package (98), the Amber99SB-ILDN forcefield (104) was employed for protein, and Amber99SB force field (91, 105,106) with modification by parmbs0 (107) was used for DNA. In addition, tovalidate the convergence of our MD simulations, we conducted another fiveindependent production simulations by GROMACS 5.0.4 (108, 109). Thus, atotal of 10 50-ns product MD trajectories were used for our data analysis(SI Appendix, Fig. S11).

To further determine the roles for critical amino acid residues that stabilizethe local structure of the active site, several classical MD simulations formutants of reactant and first pentacovalent phosphorane intermediate (i.e.,INT1) were carried out. As the intermediate contains five covalent bonds, anAmber-compatible force field has to be developed to simulate this state. Anew residue (INT1; Fig. 1 B and D) composed of the phosphated nucleotideT10′ of target DNA strand was defined. This residue (INT1; Fig. 1 B and D)contained six new atom types for amber force field of MD simulations: thepentacovalent phosphorus of nucleotide T10′, two hydroxyl groups con-nected to pentacovalent phosphorus, and the 3′-hydroxyl oxygen atom ofnucleotide C11′ that connects to the pentacovalent phosphorus. The ge-ometries, topology assignments, and partial charge parameters (Tables S3and S4) were characterized on the basis of the QM/MM-MD calculations forthe INT1 state of unmodified TtAgo complex (78, 110). The force constantswere chosen to be large enough to maintain the bond lengths, bond angles,and dihedral angles in the INT1 state of unmodified TtAgo complex (78,110). All MD protocols for mutants were the same as classical MD for un-modified TtAgo complex with guide and 19-mer target DNA. In total, 10 in-dependent 50-ns production MD trajectories were collected for each mutant(SI Appendix, Figs. S12–S14).

QM/MM Simulation. The initial structure for QM/MM calculations was asnapshot chosen from one 50-ns production MD simulation trajectory forunmodified TtAgo complex with guide and 19-mer target DNA. The choiceof QM subsystem is usually based on the proposed reaction schemes, andincludes fragments directly participating in the reaction (111). The QMsubsystem of TtAgo system includes the nucleotide bases T10′ and C11′ oftarget DNA, the nucleophilic water, and two Mg2+ ions treated by B3LYP(112–114) functional with 6–31G(d) (115–117) basis set. The QM/MM in-terface was described by the improved pseudobond approach (118, 119). Allother atoms were described by the same molecular mechanical force fieldused in the classical MD simulations. In our QM/MM calculations, the recentlydeveloped periodic boundary condition with Ewald method was applied toreliably describe the long-range interactions and dynamics (56, 120–122).The 12-Å cutoff was used for vdW and short-range PME interactions, andthere was no cutoff for electrostatic interactions between QM and MM re-gions. The selected initial structure was first minimized and then employedto map out the minimal energy path for the investigated mechanisms byusing the reaction coordinate driving method (78, 123). For each of the430 determined structures selected along the path, an 800-ps MD simulationwith MM force field was carried out to equilibrate the MM subsystem withthe QM subsystem being frozen. Finally, the resulting snapshot was used asthe starting structure for Born–Oppenheimer QM/MM MD simulation withumbrella sampling (65, 75, 76) that applied a harmonic potential to constrainthe reaction coordinate at the successive values. To ensure sufficient overlapbetween the successive windows, a force constant of 150 kcal·mol−1·Å−2 wasemployed for each window. Each of the 430 windows was simulated for atleast 15 ps, and the active site dynamics and those of the surroundings were

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simulated on an equal footing. The potential of mean force (PMF) wasobtained from the probability distributions along a reaction coordinate byusing the weighted histogram analysis method (124–126). The statistical errorof the calculated free energy change was estimated by computing the aver-age deviation between the calculated free energy change using half ofsampling data for each umbrella window (5–10 ps or 10–15 ps) and the freeenergy change calculated using data from the whole sampling period foreach umbrella window (5–15 ps). The first 5-ps simulation was considered asequilibration for each umbrella window. All aiQM/MM calculations wereperformed with modified Q-Chem (127) and Amber 12 (98) programs (56).

In our aiQM/MM-MD simulations for the substrate-assistedmechanism, weincluded only the nucleotides and nucleophilic water that directly co-ordinated with the Mg ions in the QM region. We did not include the co-ordinated ligands that are not involved in the bond forming or breaking ofthe cleavage reaction, such as the catalytic triad ASP residues D478, D546, andD660. This is because our aiQM/MM-MD calculations would become quiteexpensive and less effective when the QM region becomes large. In addition,in the aiQM/MM studies of enzymatic reactions with transition metal atomMg, it is quite common to include only ligands that directly participate in thereaction to save the computational cost because MM can describe suchclassical electrostatics interactions (64, 78, 128).

Protein Expression and Purification. The gene encoding full-length TtAgo wasinserted into a sumo-PET vector (Invitrogen) with N-terminal His6-SUMO tagfollowing an ubiquitin-like protease (ULP1) cleavage site. Recombinantprotein was overexpressed in Escherichia coli Rosetta 2 (DE3; Novagen) strainin lysogeny broth medium. The cells were grown at 37 °C until OD600reached 0.6 and then induced with 0.1 mM isopropyl β-D-1-thiogalactopyr-anoside at 18 °C for 12 h. Cell pellets were resuspended in buffer A (20 mMTris·HCl, pH 7.5, 0.5 M NaCl, and 2 mM MgCl2) and then lysed by French pressand centrifuged at 39,191 × g (Beckman Coulter, Avanti J-26 XP Centrifuge,Rotor ID: 25.50) for 40 min at 4 °C. The supernatant containing TtAgo wasloaded to a 10-mL HisTrap Fast Flow column (GE Healthcare) preequilibrated

in buffer A and eluted with buffer A supplemented with 200 mM imidazole. TheHis6-SUMO tag was removed by ULP1 and during dialysis against buffer A. TheTtAgo protein was further purified by HisTrap Fast Flow column preequilibratedwith buffer A. The purified TtAgo protein was concentrated to 25 mg/mL inbuffer A, snap-frozen in liquid nitrogen, and stored at −80 °C.

In Vitro Cleavage Assays of TtAgo. The 5′-phosphorylated (5′-phos-TGAGGTAGTAGGTTGTATAGT) 21-base DNA guide and 5′-Cys–labeled (5′-Cys-AATTAACCAAATATCAATATACAACCTACT ACCTCAGT-3′) 38-nt DNAtarget with the complementary sequence to the guide strand were pur-chased from Sangon Biotech. The cleavage reaction was performed bymixing TtAgo protein (1.0 μM) and guide DNA at the molar ratio of 1:1 andincubated in buffer (10 mM Hepes-KOH, pH 7.5, 150 mM NaCl, 5 mM MgCl2)for 30 min at 42 °C in a final volume of 10 μL. The reaction buffer Hepes-KOH, pH 7.5, changed with Mes-NaOH, pH 5.0–6.5, Tris·HCl, pH 7.0–9.0, inpH-dependent cleavage reaction. Next, 1.0 μM 5′-Cy3–labeled DNA targetwas added and incubated at 60 °C for the indicated times. Reactions wereterminated by addition of an equal volume of stop solution containing 8 Murea and 50 mM EDTA. The cleavage products were heated for 15 min at95 °C, resolved on 20% denaturing polyacrylamide gels, and visualized byMulti Green using FluorChem M (ProteinSimple).

ACKNOWLEDGMENTS. This work was supported by Hong Kong ResearchGrant Council (Hong Kong University of Science and Technology) GrantsC6009-15G, 16318816, 16302214, AoE/P-705/16, and T31-605/18-W; KingAbdullah University of Science and Technology (KAUST) Office of SponsoredResearch (OSR) Grant OSR-2016-CRG5-3007; Innovation and TechnologyCommission Grants ITCPD/17-9 and ITC-CNERC14SC01; National Natural Sci-ence Foundation of China Grants 31725008, 31571335, and 31630015; andNational Institutes of Health Grant R35-GM127040. This research made useof the resources of the Supercomputing Laboratory at KAUST. X.H. is thePadma Harilela Associate Professor of Science.

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