Visualizing Phosphodiester-Bond Hydrolysis by an Endonuclease

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Visualizing phosphodiester-bond hydrolysis by an endonuclease

Article in Nature Structural & Molecular Biology · December 2014 Impact Factor: 13.31 · DOI: 10.1038/nsmb.2932 · Source: PubMed

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Rafael Molina Stefano Stella Spanish National Research Council University of Copenhagen

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All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Rafael Molina letting you access and read them immediately. Retrieved on: 13 July 2016 © 2014 Nature America, Inc. All rights reserved. Switzerland. Correspondence should Switzerland. Correspondence be addressed to G.M. ( Barcelona, Barcelona, Spain. Biology,Computational Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain. Copenhagen, Denmark. 2 1 higher their of because but DNA, hydrolyze enzymes, restriction inteins or proteins) host as self-splicing with fusions (as introns within genes as freestanding either encoded this of reaction. understanding crucial molecular to the observed hampered has been this and never date, has endonuclease an by generation DSB of DNA course the However, and to knowledge, our states. catalytic final initial the of snapshots providing metals, divalent different of hydrolysis bonds prevent or phosphodiester of permit to forms, the in substrate-bound and endonucleases struc apo different of and structures kinetic by Crystal studies. analyzed tural been has cleavage 3 a Endonuclease with ends DNA are reaction the phosphorus of the at configuration of inversion to nism ions metal divalent require to shown been has bonds phosphodiester of Hydrolysis more-efficient of applications. development of the any hindering for thus established enzymes, be these to yet has cleavage DNA of mechanism the to led sequences, DNA technologies of recombinant-DNA blooming certain cleave specifically can which phos of breakage enzymes, and in of the 1970s restriction The discovery bonds. phodiester formation the All encompass nucleases. by processes degradation these and factors various by editing and to repair subject by polymerases, is synthesis including processes many chemical information, genetic encoding molecule key a DNA, residues, its has of different endonuclease been The Jesús Prieto Rafael Molina endonuclease Visualizing phosphodiester-bond hydrolysis by an nature structural & molecular biology molecular & structural nature Received 19 September; accepted 12 November; published online 8 December 2014; Macromolecular Crystallography Group, Crystallography Macromolecular Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Group, Crystallography Macromolecular Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Homing endonucleases are a related collection of enzymes enzymes of collection related a are endonucleases Homing

two position

a enzymatic 4

observed. crucial , which is suggested to occur in a concerted fashion leading leading fashion concerted a in occur to suggested is which ,

metals

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once

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involved I-DmoI, & MontoyaGuillermo

hydrolysis Here 1

the , 5

triggering steps, 3 , , Stefano Stella Joint Joint Barcelona Computing Center (BSC)-Centre for Genomic Regulation for (CRG)-Institute Research in Biomedicine (IRB) Program in

DSB

we and

trapping 5 in

These These authors contributed equally to this work. 7

and follow , have been determined in the presence presence the in determined been have ,

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Present Present address: Department of Chemistry and Biochemistry, University of Bern, Bern, targeted

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widely by have added electronic detail to the reaction mechanism by using using by mechanism calculations. theoretical reaction the to detail electronic added have ( reaction the of view a unique providing mechanism, catalytic this with associated ments ele transient and changes structural crystal the here X-ray by Wereport lography. reaction DNA-cleavage the of course the watch polymerases DNA for shown previously as I-DmoI–DNA in intermediates, rate catalytic the capturing reaction crystals, the down slow to method a oped metal- three display endonuclease centers catalytic sites binding whose members family ions metal the divalent key coordinate motifs LAGLIDADG I-DmoI of end the at located target asymmetric enzymes cut restriction II type to 22-bp double-strand a staggered a recognizes generating it sequence, and endo family, homing LAGLIDADG nuclease the to belongs enzyme monomeric archaeon the of endonuclease intron-encoded targeting gene specific for variants to templates are generate suitable enzymes that these thus suggesting targets original their than other sequences DNA recognize to LAGLIDADG sev the of and members families, eral structural five into Homing bp. classified are 45 to 14 endonucleases from ranging within sequences DNA locations at single genome, even a or few in occur cuts their specificity

do the cleavage Here we set out to study the catalysis of this enzyme. Wedevel enzyme. this of catalysis the study to out set we Here

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4 Departament Departament de Bioquimica, Facultat de Biologia, University of

(DSB) mechanism. , , María José Marcaida

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10– 3 ,

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© 2014 Nature America, Inc. All rights reserved. The enzyme contained in crystals grown at 15–20 °C is inefficient inefficient is °C 15–20 at grown crystals in contained enzyme (ref. The °C 88 at point melting a shows protein the Indeed, tures. at tempera high occurs that I-DmoI’s thus implying activity optimal reach up to 90 °C in an environment acidic mobilis D. Phosphodiester-bond RESULTS s e l c i t r a according to the order of cation entrance. cation of order the to according C) B and A, (site labeled alphabetically are sites metal-binding different The lines. dashed as (PDB 2 the with superimposed site active I-DmoI the ( reaction. DSB DNA the and complex protein–DNA crystallized the in intermediates reaction the capture to ( shown. are measurements three of s.d. with Averages 6.0. pH at measured activity ( below. depicted is target DNA I-DmoI The respectively. states, cleaved and noncleaved the represent states plasmid linearized and supercoiled The °C. 65 at measured reaction DSB I-DmoI the of dependence ( reaction. catalytic I-DmoI the of 1 Figure No. atoms R No. reflections Resolution (Å) Refinement Redundancy Completeness (%) I R Resolution (Å) Cell dimensions Space group Data collection Table 1 Supplementary Table 2 One crystalwasused foreachdataset.Values inparenthesesareforhighest-resolution shell.Ramachandranstatistics,wavelength ofdatacollectionandtemperatureare in r.m.s. deviations B c b /

F factors work merge ) Schemes of the procedure followed followed procedure the of ) Schemes ) Temperature-dependent I-DmoI cleavage cleavage I-DmoI ) Temperature-dependent Bond Bond angles (°) Bond lengths (Å) Water Ion DNA Protein Water Ion / active site ion DNA Protein α a σ o , , – , , I b / β , , 2VS , , F R c γ c free (Å) (°) electron density contoured at 1.2 at contoured density electron

pH and temperature dependence dependence temperature and pH Data Data collection and refinement statistics 7 optimal growth temperatures can can temperatures growth optimal ) 2 1 . Metal-ion coordination is shown shown is coordination . Metal-ion . GS,groundstate;RS, roundstate;NS,nickedtransitionstate. d ) Detailed view of view ) Detailed 90.0, 90.0, 119.89, 90.0

catalysis 106.57, 106.57, 70.35, (2.85–2.70) 46.20–2.70 46.20–2.70 99.7 99.7 (99.5) 0.07 (0.64) 0.18 0.18 / 0.23 12.5 12.5 (2.0) 4.6 4.6 (4.4) 0 h(GS) 37,807 106.60 1.192 0.009 3,057 4,625 2.70 58.9 85.3 70.1 62.5 3 3 / 3 P 98

2 a 1 ) ) pH

crystals

1 90.0, 90.0, 119.55, 90.0 7 , 107.19, 107.19, 70.85, (2.74–2.60) 46.64–2.60 46.64–2.60 97.3 97.3 (95.6) 0.07 (0.65) 0.18 0.18 / 0.22 15.4 15.4 (2.4) 4.9 4.9 (4.8) 42,060 107.23 1.212 0.010 3,057 4,636 50.1 41.3 57.8 51.7 2.60 6 6 / 6 a b 169 P Supercoiled 1 h

% cleaved product 3´ 5´

Linearized 2 100 –1 20 40 60 80 1 2 0 Nicked I-Dmo –1 – 1 5 0 pH 10

–9 + I advance online publication online advance –8 – –7 5 10 –6 35 90.0, 90.0, 119.58, 90.0 –5 107.04, 107.04, 70.66, 2 ° C – –4 7 (2.85–2.73) 15 42.86–2.73 42.86–2.73 99.9 99.9 (99.9) 0.11 (0.57) 0.18 0.18 / 0.22 10.5 10.5 (2.3) ). ). 6 4.7 4.7 (4.6) –3 - 8 h(RS) 36,886 107.27 + 1.284 0.010 3,057 4,622 10 10 / 9 40.4 42.0 51.1 44.1 2.73 –2 108 P 40 20

–1 2 I-DmoI at different pHs and found that acidic pH affected the the affected pH acidic that found and pHs different at I-DmoI Weassayed reaction. the of energy activation the overcome cannot at ° – 1 Time (h) C 2 1 7

25 cleaving the DNA target because at this temperature the protein protein the temperature this at because target DNA the cleaving + 4 3 Strand Bnoncodingstran 50

30 –

° Strand Acodingstran 8 C 6 5 + 35 90.0, 90.0, 119.83, 90.0 8 7 – 106.52, 106.52, 70.15, 65 9 40 (2.42–2.30) 46.29–2.30 46.29–2.30 96.7 96.7 (79.9) 0.05 (0.48) 0.19 0.19 / 0.22 ° 1 9 16.0 16.0 (2.0) + C 4.4 4.4 (3.0) 2 d(NS) 58,978 106.70 1.194 0.010 3,060 4,521 1 0 52.6 49.6 64.2 54.8 2.30 9 9 / 9 119 P 45 1 1

2 1 1 2 nature structural & molecular biology molecular & structural nature c d d 50 3 state (0h) 5´ 3´ Ground Time

d 2

O O

N129 P

Catalytic water Noncoding strand 90.0, 90.0, 119.62, 90.0 –2C +M 107.13, 107.13, 70.67, P –3C (2.69–2.55) 46.56–2.55 46.56–2.55 n 96.6 96.6 (94.8) 0.05 (0.54) 0.17 0.17 / 0.22 H 17.2 17.2 (2.8) 2+ 6 d(DSB) 4.9 4.9 (4.8) O 2 E117 O 44,139 107.38 1.167 0.009 3,063 4,547 10 10 / 9 62.9 53.0 68.1 58.0 2.55 144 P

2 H DSB reaction 1 +

OH O B – P A116 G2 Water

C 0

P Coding strand OH K120 O D21 – A H 90.0, 90.0, 119.77, 90.0 + Divalent meta 106.80, 106.80, 70.41, (2.42–2.30) 46.27–2.30 46.27–2.30 95.9 95.9 (92.2) 0.05 (0.50) 0.18 0.18 / 0.22 13.5 13.5 (2.7) 3.9 3.9 (3.8) 107.17 58 58 929

1.144 0.009 3,063 4,588 H O 52.4 45.2 85.8 52.3 2.30 9 9 / 9 OH 118 P P 8 d 3G 2A

Final cleave 2 I-Dmo 1 l state Q4 OH P I 2

© 2014 Nature America, Inc. All rights reserved. A water molecule occupies a second metal site (site B)The in state 1 ( ( molecules ing the DNA strand), D21, the carbonyl group of A116 and two water from DNA (2A strands previously been reported has as center, active I-DmoI the in metal divalent only one indicated which to according its signal, anomalous assigned and ground state of the reaction and thus represents the time defines 0 (statestructure This 1, catalysis. of stages the all during occupied fully is A site center. metal active the Therefore, the in present cation (site A); this site displayed the same conformation independently of the Zn as refined ion, I-DmoI–DNA crystals in the absence of catalytic catalysis metals, hinders a but divalent metal Ca of presence the in crystals the ground state of the endonuclease catalysis, we grew I-DmoI–DNA To prevent phosphodiester hydrolysis and to provide a first glimpse of The the entrance and exit of cations during the reaction. phosphodiester bonds. We used the anomalous signal of Mn 2 ( this procedure and collected diffraction data to a resolution of 2.0–2.7 WeÅ intermediatesK.different followingreactionby 77 seven trapped ates by freezing specimens in liquid nitrogen at intervals and capturing the reaction intermedi their corre and crystals multiple freezing by collecting d, 10 during reaction the of course the lowed organization ( ence of Mg pres the in structures and reaction, cleavage Mn of use The ( metals the locatehelping to through its anomalous diffraction signal, thus detection Mn crystallographic allows cation This mM 5 containing liquor ions into a preequilibrated solution of mother grownring crystals in the absence of catalytic (ref. 26). We triggered the reaction by transfer DNApolymerase with study a in observed related to packing,crystal and it has also been the reaction time course of delay This Methods). (Online d 10 in ops whereas the complete catalysis in crystals devel this temperature and pH elapses in 3 d ( conditions at 40 °C. The reaction in these solution at fulfilling crystals suitable obtained we catalysis and yielded good diffraction. Finally, at different temperatures were compatible with we examined whether crystals grown at pH 6.0 because crystals grew only at pH ( 6.0. Therefore, reaction the down limited in the use of pH in our attempts to slow ( 5 atpH decrease strong a displayed which activity, enzymatic nature structural & molecular biology molecular & structural nature displaces cation second a reaction, the of progression the during but Tables1 F o – 3

arrival initial F a c ). In both cases, the presence of the metal was unambiguously unambiguously was metal the of presence the cases, both In ). and 2 1 and . At this stage, the metal atom is coordinated with phosphates s 2+

ponding data sets at different time catalytic

Fig. Fig. 1 of F showed an identical active-center o

Supplementary Fig. 2 2

– a ). We). monitored reactionprocessthe byfollowing the 2+

F second d c did not seem to alter the the alter to seem not did 2+ Fig. 1 Fig. electron density maps showing the disruption of the and , was always found in the same metal site as Ca as site metal same the in found always was ,

upeetr Fg 1 Fig. Supplementary 2 state 1 ( strandA Supplementary Movie 1 Supplementary in crystallo a

Fig. 1 Fig. metal ). However, ). we were

of Tables1

and −3C I-DmoI 2+ d

in 2+ ). Although we attempted to obtain obtain to attempted we Although ). , a cation that allows DNA binding binding DNA allows that cation a , (

is probably the ). We fol i. 1 Fig. Fig.

and strandB active

b 2 c η ). ). ). ), - - - - - ) ; with subscripts indicat subscripts ; with

site

Table 2 statistics, wavelengthofdatacollectionandtemperaturearein One crystalwasusedforeachdataset.Values inparenthesesareforhighest-resolutionshell.Ramachandran r.m.s. deviations B No. atoms R No. reflections Resolution (Å) Refinement Redundancy Completeness (%) I R Resolution (Å) Cell dimensions Space group Data collection ). / factors work merge advance online publication online advance Bond Bond angles (°) Bond lengths (Å) Water Ion DNA Protein Water Ion / active site ion DNA Protein α a σ , , , , I b / β , , , , R c γ free (Å) (°)

Data Data collection and refinement statistics 2+ to follow Fig. Figs.

d 2a 2+ ), -

46.49–2.40 46.49–2.40 (2.53–2.40) 106.98, 106.98, 70.39, 107.15 90.0, 90.0, 119.81, 90.0 ing by A Ca and B, is populated between C, (site site metal central A Metal the scissile DNA molecule or a network of solvent molecules solvent of network a or DNA molecule scissile the pathway, an alternative one i.e., the groups phosphate involving from should of follow water activation the molecules catalytic nucleophilic usually found in contact distance with metal-bound waters endonucleases in that hydrolytic residues typically acting as other base catalysts areto not compared unique are family LAGLIDADG the tonated divalent ions exhibit a shifted p 2a Figs. S in expected as products, configuration inverted the obtain to strand each in groups phosphoryl the attack could they which from are a in waters position These B, respectively. bonds. phosphodiester the of hydrolysis the plish to accom apart far A B and are too site in metals the reaction, of the ~4 other Å each from located be should cations the mechanism, to a through two-metal-ion allow catalysis canonical of in seconds process the initial the occurs reaction. approach, this reaction slowed our Evenin molecules. water two and (3G strands DNA from phosphates with coordinated is B site in atom metal The that in site A (state 2, this water molecule in site B until it reaches the same occupancy level as Two water molecules coordinate the divalent ions in sites A and and A sites in ions divalent the coordinate molecules water Two A sites to and B 8 is Å. close However,metal between distance The

99.4 99.4 (90.0) 0.06 (0.53) 0.18 0.18 / 0.23 12.3 12.3 (2.3) endonucleases 10 d(FS) 3.7 3.7 (3.6) 53,993 3,063 4,579 1.269 0.010

2.40 51.2 45.6 61.9 54.1 6 6 / 6 101 P entrance 2

2 8 and 1 . Regarding the proton acceptor, homing endonucleases of endonucleases homing acceptor, proton the Regarding .

strandA 3 a ). It is known that water molecules associated with with associated molecules water that known is It ).

in and −2C and 22–

the Figs. Figs. 2a 24

46.49–2.20 46.49–2.20 (2.32–2.20) , center 106.89, 106.89, 70.67, 107.14 3 1 90.0, 90.0, 119.80, 90.0 . In the case of I-DmoI, a water molecule molecule water a I-DmoI, of case the In . Supplementary Table 2 strandB Mg 0.06 0.06 (0.42) 0.19 0.19 / 0.22 and 99.6 99.6 (100) 13.1 13.1 (3.4) 4.1 4.1 (4.2) i. 1 Fig.

K 70,338 of 2+ 3,057 4,644 1.159 0.009 2.20 51.8 33.3 55.1 46.4 a 6 6 / 6 259 P , state2 value that makes them easily depro 3a

), E117, the carbonyl group of G20 G20 of group carbonyl the E117, ),

the 2 1 , d b 2+

active and , hc i lctd Å away, Å 4 located is which ), , , Mg N Supplementary Movie Supplementary 1 2+ 2-like reactions (state 2, 2, (state reactions 2-like . FS,finalstate.

site or Mn 46.60–2.35 46.60–2.35 (2.48–2.35) 5 106.31, 106.31, 70.27, 107.17 , 6 90.0, 90.0, 119.59, 90.0 . Thus, at this stage . at stage Thus, this s e l c i t r a 2+ Mg 98.8 98.8 (98.7) 0.07 (0.46) 0.18 0.18 / 0.22 10.6 10.6 (2.3) 3.9 3.9 (4.0) in other hom in other 56,723 2+ 3,057 4,668 1.228 0.009 2.35 49.2 45.1 57.2 47.4 6 6 / 6 167 P , state7

2 9 . . Thus, the 1 7 ,

29 , 3 0 ). ). - - -

© 2014 Nature America, Inc. All rights reserved. calculations, which indicate a decrease of ~26 and ~56 kcal/mol of of kcal/mol ~56 and ~26 of decrease a indicate which calculations, PB by supported is This repulsion. electrostatic of because C in ing C. to transferred be consequently would which B, in bound metal the replaced actually ( C and B A, in B bound ions three and with ended A trajectories of the the 8,7% only sites, in metal a of location the after the C in modeled ion an we of when binding Finally, B. in ion an placed (49.6%) lations ( A site in bound previously is metal a when C site in ion an placed simulations PELE 127 the of one Only data. structural our supporting thus transitions, with these associated kcal/mol) be (~15–20 a barrier energy potential ( theory of level (QM(DFT)-MM) mechanical molecular and theory) functional sity (den mechanical quantum combined the at performed calculations pass through one of these sites ( ions the trajectories effective of the 85% than more or B. Actually, in A sites exposed more the in the trapped be if to assumed is ion explained incoming be can observed, experimentally as site, occupied last the is C that fact The occupancy. ion of sequence the explaining the to ( accessible solvent more therefore are and at opposite sites in the entrances of this channel of access for the ions. Sites A and B are located by the protein–DNA complex is the main route formed channel the that suggest calculations and Methods (Online site active the into ions of entrance (PELE) exploration last Monte the the Carlo–based protein energy landscape used We being site. cation-binding despite occupied ions divalent for affinity high a displays C site that indicate 1 Table state 3 Methods (Online and of simulation (MD) dynamics molecular a metal, using a large number of structures from the with energies interaction the compute to (CMIP) potential interaction molecular classical (PB) Poisson-Boltzmann 4 (state3, position central the ion that enters a metal third indicating result a reaction, the of course the during C site at catalysis grew that a signal anomalous metal observed of final and states ground the in site this occupies reaction. the of course the during strands coding and noncoding the of bonds phosphodiester the at density electron ( work. this in captured intermediates reaction structural different seven the displays course time reaction The 5 at contoured is density maps’ omit The structures. refined corresponding their onto with ( reaction. hydrolysis phosphodiester 2 Figure s e l c i t r a the in energy interaction site C when Mn a

a ) Detailed views of the active center, center, active the of views ) Detailed Therefore, Therefore, fast in binding sites A and B bind would disfavor metal and and F o – –

Supplementary Movie 1 Movie Supplementary F ). Interestingly, these calculations calculations these Interestingly, ). Structural time course of the the of course time Structural c(0 d–10 d) d–10 c(0 Supplementary Fig. 3g Fig. Supplementary upeetr Fg 4a Fig. Supplementary Supplementary Fig. 3 Fig. Supplementary Supplementary Fig. 4c Fig. Supplementary Supplementary Fig. 3e Fig. Supplementary omit maps superimposed superimposed maps omit 3 2 b 3 1 method to map the the map to method ) Plot of the 2 the of ) Plot . Nevertheless, we we Nevertheless, . Figs. 2a Figs. Supplementary Supplementary 3 2 Supplementary Fig. Supplementary 3a calculations calculations

, ). We used used We ). h F ). Interestingly, the incoming ion ion incoming the Interestingly, ). ,

b o , ). PELE PELE ). 3a 3a – ), thus thus ), σ . F ,

d 2+

c and ,

) indicate that there would would there that indicate ) f ions are previously bound ), whereas 63 other simu other 63 whereas ),

–3C a –2C , c ). ). Additional Noncoding strand advance online publication online advance Ground state Nicked state Final state B State 1 State State 10 d 2 d 0 h C 4 7 A - - - of cleavage. of catalysis. of order sequential mechanism the for responsible be to seem not However, does it two-metal-ion classical the in site second the metal of that to equivalent role a has site central the in metal this Thus, target. DNA the two-metal-ion of digestion for the triggering geometry and catalysis proper the two allowing the reaction, the in between site role pivotal a has C site in ion active metal third The reactions. the hydrolysis of rearrangement formational strands DNA the family of the cleavage of sequential members monomeric other and rvos tde hv sgetd ht N cevg i I-DmoI in cleavage DNA that suggested have studies Previous Noncoding-strand binding. cation of terms in frustration of paradigm the ( occupancy metal lower displayed that site the also but reaction the during pied occu site metal last the C is not only that showing observations, our with agreement in are results theoretical These catalysis. for cations ( possible still is Nevertheless, PB and PELE calculations indicate that binding at site C ( respectively A B and sites, to A or to both 2A Furthermore, this metal-binding site should this metal-binding Furthermore, have important impli 3G Catalytic water b

Fig. 3a Fig. 2Fo–Fc electron density of the phosphodiester bonds (σ) Metal entranceinsite 0 1 2 3 4 Supplementary Figs. 3g Figs. Supplementary Double-strand break 1 Coding strand

, hydrolysis b nature structural & molecular biology molecular & structural nature State 5 State ) 1 h 6 d and suggesting that the enzyme works under under works enzyme the that suggesting and Noncoding strand

2 cleaved

and B 1 0 Water

log (time(h)) nicked , 2 h 3 d Divalent metal and and Supplementary Table 1 Supplementary

4 state tu icuig con a including thus , Metal entranceinsite Metal exitinsite 100 4b 2 ). State 4 State 6 8 d should involve involve should 8 h

6 3 Coding strand Mn cleaved C 2+ ion 1,000 C 2 ). ). 1 - - -

© 2014 Nature America, Inc. All rights reserved. of the noncoding-strand phosphodiester bond. ( bond. phosphodiester noncoding-strand the of ( cleavage. phosphodiester-bond of absence the in metals three the of allocation the 4 Figure suggested previously that to compared order reversed a suggesting reaction. hydrolysis the in nucleophile the as role its confirms group leaving alkoxide the of face opposite the in present initially molecule water typical of the pearance a in expected S as atom, phorous by phos the at characterized configuration of inversion is the I-DmoI of structure nicked The process. catalytic global the in ( bonds O3 both with metal interacts The strands. both in reaction (TSs) the of states stabilizing transition in charged critical highly is the C in cation ( The 77% at cleaved fully is and C in occupancy 58% with hydrolyzed be to starts −2C (2A bonds phosphodiester scissile (3G −3C strands DNA from phates phos with coordinated is C in atom metal and 4, (state strand noncoding the in −2C the between to hydrolysis coupled is phosphodiester of ion initiation the metal third a by C site reaction. the of course the during C sites B and A, the of occupancy Mn of 4 at contoured is density 6 at contoured density show maps anomalous All generation. DSB after site central the in ion metal crucial the of exit the and C, B and A, sites in cations the of entrance sequential the displaying intermediates, reaction seven the of maps ( catalysis. during hydrolysis 3 Figure nature structural & molecular biology molecular & structural nature angstroms. in lines dashed with N Our data unveil the sequential cleavage of this endonuclease, endonuclease, this of cleavage sequential the unveil data Our in molecule water a of substitution The 2-type mechanism. Moreover, the disap the Moreover, mechanism. 2-type 3 strandB strandB 2+ a and and in site C. ( C. site in

i. 4 Fig. Detailed view of the active site showing intermediates of the hydrolytic reaction. ( reaction. hydrolytic the of intermediates showing site active the of view Detailed Metal ions and phosphodiester-bond phosphodiester-bond and ions Metal a ), D21, E117 and O3 and E117 D21, ), ). The first phosphodiester bond bond phosphodiester first The ). K120 Supplementary Movie 1 Movie Supplementary σ b except in state 3, for which which for 3, state in except ), playing an essential part part essential an playing ), strandB b ) Chart showing the metal metal the showing ) Chart –3C Q42 A116 σ ′ 2. and −3C and 2. atoms of the scissile scissile the of atoms to show the entrance entrance the show to 7 0 2. Metal insiteC 5 A a 2. ) ) Mn 2 State 3 2. 2. 2 1 D21 2. 3. 6 7 ′ 2+ C 2. atoms of the of the atoms 3 strandB anomalous anomalous 2. strandA –2C 1 strandA Figs. 2a Figs. 2A 2. 3. E117 Fig. 3 Fig. 7 8 2. 2. I-Dmo 0 1 ). The The ). bases bases B 2. and and 3 and 2. G20 2. I b 0 8 2. , N129 ). ). b 5 c - -

) Final hydrolytic state depicting the DSB and the metals in sites A, B and C. All distances are shown shown are distances All C. B and A, sites in metals the and DSB the depicting state hydrolytic ) Final Noncoding strand

3G a advance online publication online advance b –3C –2C K120 State Final state State 7 State 1 Nicked state Ground state 10d B Q42 Catalytic wate 4 2 d 0 A116 –3C 1. C State 4 h 2. 9 Noncoding strand 1 A 2. Nicked state 4 2.2 A 2. 3 D21 r 3. 2. and the absence of an equivalent amino acid in site B, together together B, site in acid amino equivalent an of absence the A and site to close (K120) I-DmoI residue in charged between positively a disturbed of is presence The symmetry symmetry this conserved however, B; a and A display sites I-CreI, as such family, data biochemical by 8 1 2A 2. 3G C 2A 2 2. 2 Coding strand 2. 3. 3 b E117 8 –2C ) View of the active site in the nicked state showing the breakage breakage the showing state nicked the in site active the of ) View b 2. 1. 2 9 Site occupancy (%) G20 B 2. 10 2.

3 10 20 40 50 60 70 80 90 30 1 State 0 0 2. 2. Coding strand a 4 1

) Zoom view of the active site in state 3, displaying displaying 3, state in site active the of view ) Zoom 0 N129

10 Metal entranceinsite 3G 0 S 1 Double-strand break Wate 20 State 2 Time (s) 0 tate State c

r 30 2 1h 0 2 1 . The dimeric members of the LAGLIDADG LAGLIDADG the of members dimeric The . 40 6 d 5 Site 0 Site Site

K120

Mn 50 Water Q42 C 0 B A 2+ 600 ion State B

2. 1 A116 –3C Double strandbreak 3 2. Noncoding strand A 1 2. 3 3 2. Divalent metal S 2. 2 cleaved 2 tate

D21 50 3. 2. 8 State 5 1 S 4 C 2A 2. 2. 2 2

2. 100 E117 3. 4 –2C Time (h) G20 9 Metal entranceinsite Coding strand 2. 2. 0 4 cleaved Metal exitinsite B 2. 3 tate State 2. Mn 2. 2.

150 State 2 s e l c i t r a 1 3 5 2+ N129 8 h 8 d ion 3G 3 6

State 200 6 C

C S

250 tate 2 7 3 .

© 2014 Nature America, Inc. All rights reserved. structures, which were reproduced only when the O3 the when only reproduced were which structures, crystal the in found Å ~3.1 of distances typical the with inconsistent ( group phosphate the from away we product hydrolyzed O3 the the that found in but QM(DFT), the at reaction less We of enzyme. site the it found a to be barrier- of catalytic the model ide bond in ion a on the group phosphate of phosphodiester a scissile O3 the of activation consequent and protonation the requiring without We O3 the considered we when reactants the to collapsed O3 structures the however, leaving protonated; be to the assumed when optimization upon structures Methods). We(Online only changes minor observed geometrical from crystal optimizations geometry QM(DFT)-MM with 5–7) (states process catalytic the of stages final the to corresponding structures the of strand. coding the in one hydrolysis second subsequently and strand noncoding the in bond catalysis, the triggering hydrolysis of the first targeted phosphodiester in role a crucial has cation central the Consequently, reaction. of the step limiting is the strand of this cleavage the that indicating thereby d, 6 needs hydrolysis second the whereas d, 2 in elapses bond ester crystallo in is coordinating the metal ions in sites B and C ( and phos configuration its of The inversion characteristic the catalysis. displays phorus of mechanism two-metal-ion classical the in site metal second the of that to equivalent role a has site central the 2a Figs. 3G 90% to up ( increases C site of occupancy The reaction. the of stage next the in occurs bond phosphodiester second the of cleavage The Coding-strand cod the of two-metal-ion strand. ing those to a comparison by in fulfilled hydrolysis better are mechanism phosphodiester the out carry to requirements catalytic the the because after ion divalent strand third the of noncoding arrival the for conducted quickly is reaction ( in K120 of catalysis relevance the supporting affected, is the strand of noncoding catalysis the how shows K120M mutant of activity catalytic reactions of bonds phosphate-sugar of intermediates disruption and involving TSs charged negatively the sta that bilize residues key Positively be to shown been hypothesis. have acids this amino charged cor supports the state, stabilize transition substantially responding would in and bond strand scissile the noncoding to close the very is an which K120, produce ( of B, cleavage presence of The site order at the determine glutamate might that bulkier asymmetry a of presence the with site. active the C abandons site in metal ( strand. coding the in hydrolysis phosphodiester second ( intermediate. nicked the generating strand noncoding the of cleavage a fast promote to area this in environment chemical the with together C cooperate A and in ( C. site in present is molecule a water time, this During cation. another ( 5 Figure s e l c i t r a protonated ( b a i. 3 Fig.

) The A metal site is initially occupied, and site B is rapidly filled with with filled rapidly B is site and occupied, initially is site A metal ) The ) The water molecule in C is displaced by a third metal ion. The metals metals The ion. metal a third by displaced C is in molecule water ) The To analyze the nature of the O3 the of nature the Toanalyze strandA ′

leaving groups. We reproduced the nucleophilic attack of a hydrox explored the possibility that I-DmoI catalyzes the reaction reaction the catalyzes I-DmoI that possibility the explored b

Schematic representation of the I-DmoI catalytic mechanism. mechanism. catalytic I-DmoI the of representation Schematic , n te hshdetr od ewe 2A between bond phosphodiester the and ), and is hydrolyzed, thus completing the target digestion (state 5, (state digestion target the thus completing is hydrolyzed, Supplementary Fig. 5 Fig. Supplementary conditions the complete cleavage of the first phosphodi first the of cleavage complete the conditions Supplementary Supplementary Fig. 7 3 c a ) The coupling of the metals in B and C accomplish the the C accomplish B and in metals the of coupling ) The

phosphodiester and and ′ atom coordinates the ions in sites B and C 3.9 Å 3.9 C and B sites in ions the coordinates atom Supplementary Movie 1 Movie Supplementary

hydrolysis ). Consequently, our results suggest ). All these data suggest that the the that suggest data these All ). ′ leaving groups, we examined the the examined we groups, leaving upeetr Fg 6 Fig. Supplementary ′ atoms to be deprotonated. deprotonated. be to atoms

and Fig. Fig. 4 ). Again, the metal in in metal the Again, ).

29 DSB , 35– d c ) The transient transient ) The ). ). Hence, in our

3 generation 7 ′ atoms were were atoms ′ . Indeed, the the Indeed, . atoms were were atoms strandA advance online publication online advance ). This is is This ). Fig. 4 Fig. and and b ). ). - - - - -

ie hi o D1 te abnl f 16 te 5 the A116, of the with carbonyl coordinated the is D21, A of site in chain ion side metal the DSB, the After The out. ruled be cannot mechanism this performed, lations the leaving O3 to hydrogen-bonded are molecules water no Although solvent. bulk option is the third that the proton be Finally from captured the could that this mechanism would catalytic be feasible in the case of I-DmoI. estimated ( measurements from kinetic kcal/mol 24.3 of barrier free-energy the with agreement ( kcal/mol 24.8 of barrier energy an We estimated before. as site active the of model same the We performed QM(DFT) calculations considering this scenario, using O3 the proton could also be transferred from the attacking water to molecule nucleases other for described as possibility, second The alternative. this supporting not thereby coordination, its partially loses C site in ion divalent the and changes, geometrical major tonation state for D21 and E117 at the initial However,steps of the reaction E117. show and D21 of such assuming optimizations a the QM(DFT)-MM be structures pro would role a such assume to didates as donor, proton HI RNAse as for act described could acid amino well-positioned a First, suggested: be can possibilities major Three proton. transferred the HI RNase and IV endonuclease for suggested previously as catalysis, the during that the O3 the that

5 c a 5 Glu11 Gly2 ′ ′ The need to protonate The need O3 Asn129 P O

P O

0 7 O metal O NH NH P O P O 2 ′ 2 atoms through the phosphate group involved in the reaction. group phosphate in the involved atoms reaction. the through O Metal O –3 + HP H O –3 OH P 3 O C OH OH ′ – 3 C H O

′ O O in P P P –2 O ′ P O P leaving groups should be protonated (to form O3 form (to protonated be should groups leaving O

C P site 3G O 38 ′ OH O – HO atoms in either or structures the crystal the MD simu O – –2 3G , P O 3 O O C 9 O

H O P P P C .

P O P 2A OH

O 2A O + H exits Transient metal - nature structural & molecular biology molecular & structural nature OH OH O P 3 P H ′ O 2 O 3 O N ′ NH O 3 P

P 8

the Lys120 Supplementary Fig. 7 Fig. Supplementary Asp2 2 . In the case of I-DmoI, the putative can putative the I-DmoI, of case the In . P

′ O 1 O P raises questions regarding the nature the of regarding questions raises Supplementary Fig. 1 Supplementary H

5 Gln42 O Ala116 O active ′ 2 H N 2 5 N ′

d b 5 5 center ′ ′ P P O O O O NH NH P O P O Atacking 2 2 –3 O O + –3 H O OH P 3 P C O OH C O ′ 3 H H H – ′ O O O O P P P P O O P P H O ), which is in good good in is which ), O 2 HO HO OH –2 – –2 3G O O O ), thus suggesting ), thus suggesting 3G C C H 39– O O P P P ′ P -phosphate of of -phosphate P P O O 2A O O 4 2A – – 1 OH OH O P P O , is that the the that is , O O HO 3 3 O H ′ ′ NH NH P P 2 O 2 2 P P O O O O H H 5 5 2 2 ′ ′ ′ N N H) H) 3 0 - - -

© 2014 Nature America, Inc. All rights reserved. Accession Accession codes. the pape the of in version available are references associated any and Methods M reaction. the been also has in polymerases ion observed metal transient a that noting worth is it Finally, would be more strand. than in in favorable the coding the noncoding hydrolysis initial stabilize and state, accordingly transition corresponding the strongly to expected is strand noncoding the in bond sile of order scis the to close K120 sequential of presence the the specifically, More hydrolysis. explain would B and A sites in ronments ( strands sequential DNA the the of cleavage triggers which C, site in cation this of entrance the procedure has captured lysis Our in time-resolved this endonuclease. cata to is promote Therefore the metal third essential two-metal-ion strands. both of hydrolysis the in collaborating reaction, the pivotal in a role has and sites catalytic two the metal between shared is central C in The ion catalysis. accomplish to environment chemical and geometry proper the provide to site metal central the transient enter must third ion a I-DmoI, of bio case the In essential mechanism. this use logical nucleases two-metal-ion of a by families Multiple cleavage of mechanism. stages different the of dissection time-resolved allowing molecule, DNA a in bonds phosphodiester 1 Movie ( by an DNAendonuclease DSB catalyzed These intermediates. allowed views consecutive us to watch and analyze the generation different of a capturing catalysis, the down to slow reaction the of properties chemical the of advantage taken freezing have flash crystal with trapped been previously have intermediates reaction catalytic and changes Conformational DISCUSSION departure. its promoting thus catalysis, of stage final the at impaired becomes site binding central the in ion divalent a of coordination the that indicate results these Altogether, ( B in ion the with only interact finally to further shift to observed is E117 7, state to corresponding structure the in QM(DFT)-MM with performed is from departure the site. central Moreover, if optimization a geometry ion the promotes E117 of displacement the that idea the supporting nity of site C for Mn affi binding the in decrease 7 a show drastic state in PB calculations B ion in site ( the toward is rotated and 7, (state cycle ion in C site at of end divalent catalytic the the the of departure the explain might rearrangements structural Some The 2a Figs. leave the central position and is substituted by a water molecule (state 6, 3G 5 the Mn E117, D21, with interacts which transient The molecule. water another and motif the with LAGLIDADG second interactions the in E117 of chain side the G20, of carbonyl similar has B site 5 in cation the whereas −3C nature structural & molecular biology molecular & structural nature numbers accession under Bank Data Protein the in ited ′ -phosphate of 3G ethods strandA 3

strandB final a ). ). Notably, there is a displacement of the E117 side chain, which and ). Our results show the course of specific hydrolysis of two two of hydrolysis specific of course the show results Our ). , and the 3 the and ,

state te hsht o 2A of phosphate the , 3a Supplementary Fig. 7d Fig. Supplementary , b Coordinates and structure factors have been depos and and strandA, r 2+ ′ . -hydroxyls of −2C of -hydroxyls (~21 kcal/mol) compared to that of state 6, thus Supplementary Movie 1 Movie Supplementary 25 the phosphate of −2C , 2 6 , albeit with a complete different role in role different a complete with , albeit Fig. Fig. Supplementary Movie 2 Movie Supplementary 5 strandA ). The different chemical envi chemical different The ). and and ′ -phosphates of −3C of -phosphates strandB Fig. Fig. Supplementary Movie 2 Movie Supplementary and a water molecule, molecule, water a and and 2A and 5 strandB ).

and advance online publication online advance Supplementary Supplementary , , the main chain strandA 42– 2+ 4 4UN 5 . Here, we we Here, . in site C, C, site in strandB , starts to to starts , ). In fact, ). In fact, Figs. 2a Figs. 7 o (0 h), h), (0 nline nline and and ). ). - - - -

19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 8. with with data processing and wrote the manuscript. manuscript. time-course experiment, helped G.M. the designed crystallographic J.P. the data, discussed performed biochemical experiments and helped to write the M.O. the theoretical calculations and supervised helped to write the manuscript. data sets. H.G. analyzed the data and performed the theoretical calculations. analysis and crystallographic refined the data. M.J.M. refined some of the and the performed the crystals biochemical experiments. performed the R.M. andR.M. S.S. performed the catalytic time-course experiment. P.R. prepared ERC-SimDNA to M.O.). Competitividad of Spain to andR.M. (JCI-2011-09308 BIO2012-32868 and Educación of Spain (SB2010-0105 to S.S.) and the Ministerio de Economia y Marie Curie ‘SMARTBREAKER’ to (2010-276953 S.S.), the Ministerio de Autónoma de toMadrid (CAM-S2010/BMD-2305 G.M.), the European Union to NNF14CC0001 G.M.), the Fundación Ramón Areces (to G.M.), the Comunidad Spain to (BFU2011-23815/BMC G.M.), the Novo Nordisk Foundation (grant This work was supported by the Ministerio de Economía y Competitividad of We thank the Swiss Light Source (SLS) and ALBA staff beamline for their support. version of the pape Note: Any Supplementary Information and Source Data files are available in the d), (10 4UN 9. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial C AU Ackn OMP

T Dalgaard, J.Z., Garrett, R.A. & Belfort, M. Purification and characterization of two of characterization and Purification M. Belfort, & R.A. Garrett, J.Z., Dalgaard, by encoded endonuclease site-specific A M. Belfort, & R.A. Garrett, J.Z., Dalgaard, Kjems, J. & Garrett, R.A. An intron in the 23S ribosomal gene of the archaebacterium R. Takeuchi, P. Redondo, I.G. Muñoz, L.E. Rosen, endonuclease homing of evolution Directed H. Zhao, & N. F.,Wen,Sun, Z., Chen, J. Ashworth, S. Arnould, Homing G. Montoya, & J. Prieto, F.J., Blanco, I.G., Munoz, M.J., Marcaida, Stoddard, B.L. Homing endonuclease structure and function. and structure endonuclease Homing B.L. Stoddard, mechanism. and function structure, of diversity Nucleases: W. Yang, Yang,W., J.Y.Lee, Nowotny,& two-Mg acids: nucleic breaking and Making M. RNA. catalytic for mechanism two-metal-ion general A J.A. Steitz, & T.A. Steitz, Gerlt, J.A., Coderre, J.A. & Mehdi, S. Oxygen chiral phosphate esters. in S.E. Halford, & R.J. Roberts, V. Pingoud, biology.molecular of workhorses the became enzymes restriction How R.J. Roberts, intron. archaeal an by encoded endonuclease site-specific thermophilic a I-DmoI, of forms intron. archaeal typical a mobilis Desulfurococcus (2011). modification. gene targeted meganucleases. engineered by recognition locus. RAG1 human endogenous the specificities. (2009). specificity. sequence altered with I-SceI specificity. cleavage (2006). 443–458 targets. DNA novel on recombination induce that endonucleases (2005). 49–95 (2010). 727–748 applications. therapeutic to basics from endonucleases: Biophys. specificity. substrate and catalysis USA Sci. Acad. Natl. Proc. 55 Harbor, Spring 1993). York, Cold New Press, Laboratory Harbor Spring (Cold 35–88 R.J.) Roberts, family. EcoRI (2009). the of endonucleases restriction USA Sci. Acad. Natl. Proc. HOR HOR 8 , 291–380 (1983). 291–380 , o ET (1 h), 4D6 wled I J. Biol. Chem. Biol. J. C N

44 O G G FI O NT 4UN , 1–93 (2011). 1–93 , t al. et (Mg t al. et gm Nucleic Acids Res. Acids Nucleic t al. et t al. et t al. et t al. et N t al. et r l RIBU . . A ent 9 oig noulae -rI eiaie wt nvl N target DNA novel with derivatives I-CreI endonuclease Homing NC n h dvln mtl o dpnec o DA laae by cleavage DNA of dependence ion metal divalent the On 2+ (8 h), oeua bss f eoem pgetsm ru C DNA C group pigmentosum xeroderma of basis Molecular opttoa rdsg o ednces DA idn and binding DNA endonuclease of redesign Computational Engineering of large numbers of highly specific homing specific highly of numbers large of Engineering oeua bss f nierd eaulae agtn of targeting meganuclease engineered of basis Molecular Nature apn ntrl eevis f oig noulae for endonucleases homing of reservoirs natural Tapping T , state 2) and and 2) state ,

IA s 269 IO . L Nature N Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. I , 28885–28892 (1994). 28885–28892 , 4UN

NTE S 441 90 102 rc Nt. cd Si USA Sci. Acad. Natl. Proc. , 6498–6502 (1993). 6498–6502 ,

, 656–659 (2006). 656–659 , , 5905–5908 (2005). 5905–5908 ,

R 34 A 318 E Mol. Cell Mol. (2 d), , 4791–4800 (2006). 4791–4800 , S Nucleases , 675–677 (1985). 675–677 , T Nucleic Acids Res. Acids Nucleic 4D6 S Nature 4UN rti Eg Ds Sel. Des. Eng. Protein N

22 (Mg es Ln, .. Lod RS & R.S. Lloyd, S.M., Linn, (eds. , 5–13 (2006). 5–13 , B

456 (6 d), . o. Biol. Mol. J. 2+

90 , 107–111 (2008). 107–111 , , state 7). state ,

, 5414–5417 (1993). 5414–5417 , 39 el Ml Lf Sci. Life Mol. Cell. http://www.nature.c 4UN s e l c i t r a

, 729–743 (2011). 729–743 , 108 Q. Rev.Biophys. Q. J. Mol. Biol. Mol. J.

C 13077–13082 , 393

(8 d), 22 Adv. Enzymol. 140–160 , 249–256 ,

. Rev. Q.

4UT

online 2+

355

om/ -ion 38 67 0 , , ,

Borrelli, W.K., Vitalis, A., Alcantara, R. & Guallar, V. PELE: protein energy landscape ep, J.L. Gelpí, B.S. Chevalier, catalytic three-metal-ion the Unraveling J. McCammon, & J. Tainer, I., Ivanov, the of activities by diesterase and mono- F.Phosphate Himo, & catalysis J.-G. Yu, R.-Z., Liao, on Observations P.H. Hippel, von & W.B. Lott, B.W., Pontius, J. Prieto, DNA Nakamura, a T., Zhao, Y., Observing Yamagata, S.H. Y., Hua, Wilson, Y.J. & & Yang, W. D.D. Watching DNA polymerase Shock, W.A., Beard, B.D., Freudenthal, Moure, C.M., Gimble, F.S. & Quiocho, F.A. The crystal structure of the gene targeting B. Chevalier, J.M. Bolduc, M.J. Marcaida, functional and structural endonucleases: Homing B.L. Stoddard, & B.S. Chevalier, y bfntoa hmn ednces ad ru I nrn piig factor. splicing Dev. intron I group and endonuclease homing bifunctional a by 105 engineering. meganuclease into insights new provides (2001). 3757–3774 1304–1311 (2005). 1304–1311 technique. based Carlo Monte novel a exploration: (2001). proteins. of simulations dynamics molecular in procedure endonuclease. 104 IV.endonuclease enzyme repair DNA the of mechanism Chem. Inorg. calculations. chemical quantum from insights P1: nuclease enzyme zinc trinuclear USA Sci. Acad. Natl. mechanism. Proc. two-divalent-metal-ion a with consistent are ribozymes hammerhead endonuclease. homing I-DmoI archaeal η wrong. from right choose polymerase Biol. specificity.site target its of origins the reveals I-SceI endonuclease homing endonuclease. homing LAGLIDADG nih it te aayt o ito/nen mobility. intron/intein of catalysts the into insight make a phosphodiester bond. phosphodiester a make , 1465–1470 (2007). 1465–1470 , (2008). 16888–16893 ,

17 334 , 2875–2888 (2003). 2875–2888 , , 685–695 (2003). 685–695 , et al. et t al. et

et al. et 49 t al. et Generation and analysis of mesophilic variants of the thermostable the of variants mesophilic of analysis and Generation Mol. Cell Mol. t al. et t al. et , 6883–6888 (2010). 6883–6888 , lsia mlclr neato ptnil: mrvd setup improved potentials: interaction molecular Classical Structural and biochemical analyses of DNA and RNA binding RNA and DNA of analyses biochemical and Structural ea-eedn DA laae ehns o te I-CreI the of mechanism cleavage DNA Metal-dependent Design, activity, and structure of a highly specific artificial specific highly a of structure and activity, Design, rsa srcue f -mI n ope wt is agt DNA target its with complex in I-DmoI of structure Crystal

, 895–905 (2002). 895–905 , 94 , 2290–2294 (1997). 2290–2294 , Nature Biochemistry Cell

J. Biol. Chem. Biol. J. 487

154 , 196–201 (2012). 196–201 , , 157–168 (2013). 157–168 ,

43 , 14015–14026 (2004). 14015–14026 , . hm Ter Comput. Theory Chem. J.

Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. 283 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. uli Ais Res. Acids Nucleic , 4364–4374 (2008). 4364–4374 , Proteins

45 advance online publication online advance 428–437 , J. Mol. J. Genes

1 , ,

44. 43. 42. 41. 40. 39. 38. 37. 36. 45. 35. 34.

ury JB, zk, . Soe A & ct, .. atr ad iulzto o a of visualization and Capture W.G. Scott, & A. Szoke, H., Szoke, J.B., Murray, crystallographic Direct D. Ringe, & G.A. Petsko, B.F., Rasmussen, X., Ding, observation structural Direct D. Ringe, & G.A. B.F.,Petsko, Rasmussen, X., Ding, cleavage phosphodiester associative the of details Atomistic G. Fels, & Elsässer,B. ribonuclease in cleavage Phosphodiester M. Klein, & M. Peraro, Dal M., Vivo, De diester phosphate the of study functional T.Density Yu, & N. Haruki, K., Makoto, RNA of mechanism Catalytic G. Hummer, & W. Yang, M., Nowotny, E., Rosta, Gómez, H. nucleophilically and Substrate-assisted L. Masgrau, & J.M. Lluch, H., Gómez, ct, .. Mra, .. Anl, .. Sodr, .. Ku, . atrn the Capturing A. Klug, & B.L. Stoddard, J.R., Arnold, J.B., Murray, W.G., Scott, óe, . Pla, . Til W, lc, .. Msru L Retaining L. Masgrau, & J.M. Lluch, W., Thiel, I., Polyak, H., Gómez, complexed I-SceI of structures F.A.Crystal Quiocho, F.S.& Gimble, C.M., Moure, ppiy etr usrt: xliig h “ls tasto” n rti dynamics. protein in Chem. transition” Bioorg. “glass the exploiting substrate: ester peptidyl a of hydrolysis elastase-catalyzed the in intermediate acyl-enzyme an of observation Biochemistry elastase. by substrate ester an of hydrolysis the in intermediate acyl-enzyme an of H. ribonuclease human in Soc. mechanism. catalytic two-metal-aided associative an via occurs H HI. RNase (2014). by hybrid RNA/DNA in mechanics/molecular RNA of hydrolysis quantum from H ribonuclease simulations. mechanics by cleavage backbone 12 transferase-T2. polypeptide:GalNAc UDP-GalNAc human by glycosyltransferases. retaining for bovine in catalysis assisted state. transition transfers C 1,4-galactosyltransferase 2065–2069 (1996). 2065–2069 structure of a catalytic RNA intermediate: the hammerhead ribozyme. X-ray and trapping lattice reconstruction. crystal holographic using complex enzyme-product RNA catalytic lcslrnfrs mcaim tde b Q/M ehd: lipopolysaccharyl- methods: QM/MM by studied mechanism glycosyltransferase cleavage DNA the along intermediates pathway. reaction of snapshots substrates: DNA nicked to , 2645–2655 (2014). 2645–2655 ,

130 , 10955–10962 (2008). 10955–10962 , et al.

34 Computational and experimental study of O-glycosylation: catalysis , 9285–9293 (1994). 9285–9293 , J. Am. Chem. Soc. Chem. Am. J. , 410–423 (2006). 410–423 , Nucleic Acids Res. Acids Nucleic nature structural & molecular biology molecular & structural nature J. Am. Chem. Soc. Chem. Am. J. Phys. Chem. Chem. Phys. Chem. Chem. Phys. Mol. Cell Mol. α 13glcoytaseae mcaitc implications mechanistic -1,3-galactosyltransferase: J. Am. Chem. Soc. Chem. Am. J.

134

5 36 α , 279–287 (2000). 279–287 , glcoe i a ooabnu ion-like oxocarbenium an via -galactose , 4743–4752 (2012). 4743–4752 , , 3287–3296 (2008). 3287–3296 ,

133 , 8934–8941 (2011). 8934–8941 ,

12 135 o. Phys. Mol. , 11081–11088 (2010). 11081–11088 , , 7053–7063 (2013). 7053–7063 , r. iml Chem. Biomol. Org.

112 . m Chem. Am. J. Science 355–364 ,

274 α - ,

© 2014 Nature America, Inc. All rights reserved. of conventional transition-state theory. free-energy barrier associated with the catalytic process, within the framework magnitude of the limiting rate of the cleavage event—were used to estimate the k concentrationsdifferentinitialI-DmoI ([I-DmoI] K concentration was determined with the equation [S] = [S] k as proposed for restriction endonucleases irradiation was quantified with ImageJ64 software ( previouslydescribed as processed were and buffer stop 6× of addition by stoppedwere reactions The 60 min for the reactions at pH 8, or between 0 and 20 d for the reactions at pH 6. reactionofthe mixtures were attaken regular intervalsrangingand 0 between and 10 mM MnCl 50 mM NaCl and 10 mM MgCl Tris,mM 10 DNAin 8,incubated the °Cwas withI-DmoIpH at 65 wild-type assays, the In sequence). target I-DmoI containingthe plasmid pGEM-T mid plas (3-kb substrate of nM 4 against nM) 25 and 1 (between concentrations described previously enzyme variable at performed was plasmid as circularized disappearanceof The measured was activity enzymatic the peratures, Kinetics. gel (Lonza). degrade the I-DmoI protein and then electrophoresed in a 4% MetaPhor agarose blue (6× stop buffer), incubated at 338 K for 15 min to allow the proteinase K to bromophenol(w/v) 0.048%proteinaseand mg/mlEDTA, K 1.5 mM 8, 95 pH ent times, ranging from 15 min to 24–48 h, by addition of 5 2 mM MnCl 65 °C) in either 5 mM Tris-HCl, pH 8, or 5 mM MES, pH 6, 150 mM NaCl and The experiment was done at four different temperatures (35 °C, 40 °C, 50 °C DNAcleavageI-DmoIassubstrateassays. andin used was crystallizationfor used digestion. target temperature-dependent and pH- Time-, PHENIX subjectedto iterative cycles of model building and refinement with Coot (I-DmoI–DNA–Ca PHASERprogram the implementedin as replacement, molecular by solved were structures The comparison. density electron and structure for parameters unit-cell identical and for the crystallographic data and structure solution are summarized in XDS accomplishedwerewith data sets, 157 were used to obtain the current data. Data processing and scaling ger catalysis, selecting the wavelength around the K absorption edge. Out of 535 we recorded anomalous signal from those crystals that contained Mn Barcelona, Spain). To follow the reaction steps and the role of the catalytic ions, Synchrotron,(ALBA XALOC Villigen,Switzerland)and (SLS, PXI beamlines at detectorsPILATUS with K 100 at crystals frozen from collected refinement. were data and building model solution, structure collection, Data time, we stopped the reaction by freezing the crystal in liquid nitrogen. ferent times ranging from 0 to 10 d at 40 °C. After the corresponding incubation liquorsolution includingMnCl mM 5 by transferring the I-DmoI–DNA crystals with a nylon loop intoPhosphodiester-bond another mother-catalysis liquor plus 5 mM MnCl promote controlled catalysis the absence of catalytic ions. The crystallization temperature was set at 40 °C to as described before described ously crystallization. Protein expression, purification, formation of protein–DNA complexes and ONLINE doi: * = ( * describing the disappearance of the DNA substrate ([S]) at constant enzyme m We considered single-turnover conditions to estimate the kinetic parameters, * and * 10.1038/nsmb.2932

2 k . All data were isomorphic in the * max 5 k To analyze the kinetic parameters of the enzyme at different tem different at enzyme the of parameters kinetic the analyze To 1 * [I-DmoI]

. max METHODS 2 in a 25- wereestimatedthe of curve-fittingby I-DmoI expression and purification were performed as previexpressionI-DmoIaspurification andwereperformed 21 , 2 2 4 with a 10- 2+ 7 4 9 . Protein–DNA complexes were obtained and crystallized crystallized and obtained wereProtein–DNA complexes . 6 0 . The search models were based on the PDB entries PDB the on based were models search The . except that the protein–DNA complexes were prepared in )/([I-DmoI] ) and µ l final reaction volume. Reactions were stopped at differ 2 2 7 . . The intensity of the bands observed upon UV-lightupon observed bands the intensityof The . 2 V in crystallo S µ 4 8 7 l final volume ( 2 (I-DmoI–DNA–Mn and Scala from the CCP4 package CCP4 the from Scala and in crystallo or at 40 °C in 10 mM MES, pH 6, 50 mM NaCl 0 + K m P 2 after crystal transfer to the same mother *). The values of . The incubationTheout . carried atwasdif 2 1 23 space group and were processed with . , The chemical reaction was initiated 5 2 . The pseudo-first-order constant Supplementary Fig. 1 0 http://r ) according to the equationaccordingthe to ) 2+ ) k 2 * values obtainedvaluesatthe * k 1 . The models were then * 0 max e sb.info.nih.gov/ij − k µ —representing the * The 25-bp DNA 25-bp The t l of 45% glycerol, . The parameters 4 8 ). Aliquots . Statistics . 2+ Tables 1 to trig 5 2 0 and V All All / ). 2 S 7 7 ------.

etry optimizations of the structures corresponding to states 6 and 7. This QM This 7. and 6 statescorrespondingstructuresto optimizationsthe of etry QM region had a charge of −2 and included 169 atoms for the QM/MM geom molecules within 15 Å of the same ion center was considered (~2,000region—allowed atoms).to move in The the active optimizations—includingAn atoms. ~8,200 toall residuessize system and the waterreduce to removed were C site in ofcatalysis. solventthe All molecules more than awayÅ25 from Mnthe 7 to 5 statescorrespondingto structures optimizations QM/MM in for points carried out with the NPT ensemble. simulationwas MD of ns 2 of run final conditions,a Within these structures. crystallographic the from variations structural large prevent to solutes the of for the first 100 ps, and a constraint of 5 kcal/mol was applied to the heavy atomsthe divalent cations) were completely free to move. The volumein which onlywas water moleculeskept (excluding constantthose involved in the coordination of gate gradient optimization steps, an equilibration MD was performed at 313 K, conjuof steps 2,000 Aftercatalysis. of 7 and 6 5, 3, 1,states correspondingto classical MM (AMBER) MD simulation steps to relax and equilibrate the systems suite ( added to neutralize the system with the LEaP module of the Amber 11 software Na 26 and atoms), ~53,300 of size final Å; 10 of distance (margin watermolecules TIP3P of box octahedral truncated a solvatedwith fully were systems The ligand. DNA the and ions divalent the of presence in procedure on the basis of the p chosen were lysine) and histidine glutamate,arginine, (aspartate, protein the calculations. Theoretical nique potential in the I-DmoI–DNA complex. Moreover,APBS used to compute the molecular interaction potentials (MIP) stantof withwere4 considered PBthein calculations. The CMIP programMn was also every 10 ps from the production MD simulation with an internal dielectric con ties for the ions were considered in the interaction-energy calculations.(electrostatic plusSnapshots Van der Waals) of Mn method, as implemented in CMIP ized the optimized TS structure. to ensure that one imaginary frequency and a suitable transition in vectorthe characterHDLCopt (P-RFO) optimizer functionrational partitioned the andcombines L-BFGS,that mizer Goldfarb-Shanno Energy minimizations were performed with the low-memory Broyden-Fletcher-model. shift charge the with boundary QM/MM the treat to used atomswere introduced for the nonbonding MM and QM/MM interactions. Hydrogen-link scheme ding retrieved from DL_POLY B3LYP/6-311+G(d,p) the with Gaussian09 in performed were calculations energy Single-pointory. B3LYP the at partition QM the in calculations dient ChemShell package modular groups were fixed to avoid major geometrical disruptions. ( deoxyriboses from nucleotides 2A and −3C and the adjacent phosphate groups molecules), water two and A116 and G20 residues of groups carbonyl E117, ions and its first coordination sphere (including side chains of residues D21 and structurecorrespondingonthe (stateandhincluded divalent3)three 8 tothe hydrolysis process in the active site of I-DmoI. The model of 110 atoms was based were modeled as Mg residues G20 and A116, and four water molecules). For efficiency, divalent ions tion sphere (including side chains of residues D21 and E117, carbonyl groups of the noncoding and coding strands, the three divalent ions and its first coordina partition included the four nucleotides involved in the hydrolytic reaction from Supplementary Fig. 7 The final snapshots final simulationsThe MD fromthese wereconsidered startingas and minimization includingprotocol equilibration classical a Wefollowed TheMonte Carlo–basedprotein landscapeenergy exploration (PELE) tech The linear Poisson-Boltzmann equation (PB) was solved by the finite element the with performed were optimizations geometry QM/MM or QM All QMgas-phase calculations were performedreproduceto phosphodiester-a 3 http://am 3 was used to study the migration of Mn ofmigration the study to used was 70 , 7 1 was used for the TS search. Both methodologies were implemented 6 7 was adopted in the QM/MM calculations, and no cutoffs werecutoffs no and calculations,QM/MM the in adopted was bermd.org 7 7 2 3 module of ChemShell. Frequency calculations were performed 68 was used to compute and visualize the surface electrostatic surface the computevisualize to and used was , K 6 2+ 9 a 6 LBG) loih ad h mcotrtv T opti TS microiterative the and algorithm (L-BFGS) ). During the geometry optimizations, the terminal methyl values predicted at pH 6 by the empirical H++ in all the QM or QM/MM calculations. 4 functional/basis set. MM energies and gradients were gradients and energies MM set. functional/basis nature structural & molecular biology molecular & structural nature 6 5 / The protonation states of the titratable residues of residues titratable the of statesprotonation The , with the AMBER force field ). 5 6 with TURBOMOLE with 3 2 , to estimate the interacting binding energy 2+ to sites A to C. The desolvation penal 2+ ions form the solvent to the tosolvent the formions 5 7 58– for the energy and gra and energy the for 6 6 . An electronic embed 6 2 /SVP 6 3 level of the of level 2+ + as a probe. ions were ions 53– 5 2+ 5 v3.1 ion ------

© 2014 Nature America, Inc. All rights reserved. 61. 60. 59. 58. 57. 56. 55. 54. 53. 52. 51. 50. 49. 48. 47. 46. tions, CMIP, APBS and PELE were plotted with VMD the continuum solvent model OBC or 48 h of CPU time. The AMBER99sbBSC0 force field unbiased simulations were performed and interrupted after 2,000 accepted steps andindependentprotein-DNA 127 the on, ininterface. Laterformedchannel Mn placing by calculations PELE very powerful to describe ligand migration in complex systems steered stochastic approach with structure-prediction methods, which makes it active site of I-DmoI. This Metropolis Monte Carlo–based program combines a nature structural & molecular biology molecular & structural nature

Stephens, P.J., Devlin, F.J., Chabalowski, C.F. & Frisch, M.J. Frisch, C.F.& F.J.,Chabalowski, P.J.,Devlin, Stephens, theories. density-functional local correct and Hartree-Fock of mixing with new A A.D. Becke, approximation exchange-energy Density-functional A.D. Becke, method. Hartree-Fock the of simplification A J.C. Slater, structure Electronic C. Kölmel, & H. Horn, M., Häser, M., Bär, R., Ahlrichs, P. Sherwood, prediction pK automating 3.0: H. A. Onufriev, & B. Aguilar, R., Anandakrishnan, algorithm clustering simple Onufriev,A & A. S. Narayanan, G., Grothaus, J., Myers, J.C. Gordon, N.P.Johnson, S.E., Halford, with endonuclease restriction EcoRI The J. Grinsted, & graphics. molecular for tools Adams, P.D. model-building Coot: K. Cowtan, & P. Emsley, A.J. McCoy, Evans, P. Scaling and assessment of data quality. W.Xds. Kabsch, and Crystallization G. Montoya, F.J.& Blanco, E., Ramos, J., P.,Prieto, Redondo, f irtoa asrto ad iclr ihos seta sn dniy functional density fields. using force spectra dichroism circular and absorption vibrational of Phys. Chem. J. behavior. asymptotic (1951). Lett. calculations on workstation computers: the program system turbomole. 632 catalysis. in problems to application its and approach modeling molecular simulations. atomistic and for structures biomolecular of preparation the and 63 macromolecules. in pKs of calculations in use for enough accurate be can macromolecules. to bacteriophage solution. structure Crystallogr. Biol. D Crystallogr. Acta (2007). 658–674 62 in I-Dmo-I 63 endonuclease homing the DNA. on target its with complex analysis diffraction X-ray preliminary , 928–938 (2006). 928–938 , (2006). 72–82 , (2007). 1017–1020 , , 1–28 (2003). 1–28 ,

162 , 165–169 (1989). 165–169 , et al. J. Phys. Chem. Phys. J. et al. et t al. et t al. et λ

DNA: kinetic studies. kinetic DNA: 98 PHENIX: a comprehensive Python-based system for macromolecular Acta Crystallogr. D Biol. Crystallogr. Biol. D Crystallogr. Acta Nucleic Acids Res. Acids Nucleic H.: a server for estimating p estimating for server a H.: , 1372–1377 (1993). 1372–1377 , Acta Crystallogr. D Biol. Crystallogr. Biol. D Crystallogr. Acta hsr rsalgahc software. crystallographic Phaser US: gnrl ups ipeetto o te QM/MM the of implementation purpose general A QUASI: Nucleic Acids Res. Acids Nucleic Phys. Rev. A Rev. Phys.

98 Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. Cryst. Biol. Struct. F Crystallogr.Sect. Acta , 11623–11627 (1994). 11623–11627 , 2+

8 38 1

ions 25 Å away from the entrance to the the to entrance the from away Å 25 ions . Outcomes from QM or QM-MM calcula

60 40 , 3098–3100 (1988). 3098–3100 , Biochem. J. Biochem.

, 2126–2132 (2004). 2126–2132 , , W537–W541 (2012). W537–W541 , 33 , W368–W371 (2005). W368–W371 , K Acta Crystallogr. D Biol. Crystallogr. a s and adding missing hydrogens missing adding and s

191

66 8

79 66 2 . o. tut Theochem. Struct. Mol. J. , 581–592 (1980). 581–592 , , 125–132 (2010). 125–132 , . pl Crystallogr. Appl. J. . , Phys. Rev. Phys. 8 , 213–221 (2010). 213–221 , 0 was considered with Ab initio Ab 74– 7

8 81 . We started Chem. Phys. calculation , 385–390 , Proteins

40 - ,

74. 73. 72. 71. 70. 69. 68. 67. 66. 65. 64. 63. 62. 82. 81. 80. 79. 78. 77. 76. 75.

Hernández-Ortega, A. of Electrostatics J.A. McCammon, & M.J. Holst, S., Joseph, D., Sept, N.A., Baker, and optimisation geometry scaling Linear W. Thiel, & A.J. Turner, S.R., Billeter, states. transition of location the for algorithm An J. Baker, on points stationary for Search R. Shepard, & J. Simons, N., Adams, A., Banerjee, storage. limited with matrices quasi-Newton Updating J. Nocedal, mechanical quantum combined P. for Sherwood, models Hybrid T. Walter, & B. Dirk, molecular parallel J.W.Scott, general-purpose a DL_POLY_2.0: T. Forester, & W. Smith, diffuse P.V.R. Schleyer, Efficient & G.W. Spitznagel, J., Chandrasekhar, T., Clark, sets basis Gaussian contracted optimized Fully R. Ahlrichs, & H. Schäfer,Horn, A., correlation-energy Colle-Salvetti the of Development Parr,R.G. Yang,W.& C., Lee, Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. molecular visual VMD: K. Schulten, & A. W.,Dalke, Humphrey, large- and states native protein Exploring D.A. Case, & D. Bashford, A., Onufriev, A. Pérez, V. Hornak, a globins: in pathways ligand F. Spyrakis, multiple vs. Single V. Guallar, & M.F. Lucas, M.F.Lucas, Guallar,& V. monoxide carbon hemoglobin human on view atomistic An A. Hosseini, J. oxidase. aryl-alcohol fungal on study computational and experimental an USA ribosome. the and microtubules to application nanosystems: coordinates. internal delocalised Phys. Chem. hybrid in search state transition (1986). 385–395 surfaces. 35 methods. cluster embedded approaches. (1996). mechanical molecular and proteins. and acids package. simulation dynamics set Li–F. basis elements, 3–21+G first-row for The III. calculations. anion for sets basis function-augmented Kr. to Li atoms for (1988). 785–789 density.electron the of functional a into formula Graph. (2004). 383–394 model. born generalized modified a with changes conformational scale conformers. alpha/gamma (2007). of description the parameters. backbone protein improved AHb2. (2013). and AHb1 non-symbiotic view. computational processes. migration B Chem. Phys. J.

436 , 773–782 (1980). 773–782 ,

, 341–350 (2011). 341–350 , 14 , 10037–10041 (2001). 10037–10041 , J. Phys. Chem. Phys. J. et al. et , 33–38 (1996). 33–38 , t al. et et al. et

t al. et et al. et 2 t al. et , 2177–2186 (2000). 2177–2186 , Refinement of the AMBER force field for nucleic acids: improving improving acids: nucleic for field force AMBER the of Refinement A new force field for molecular mechanical simulation of nucleic of simulation mechanical molecular for field force new A Comparison of multiple Amber force fields and development of development and fields force Amber multiple of Comparison ( Atomic picture of ligand migration in toluene 4-monooxygenase. toluene in migration ligand of picture Atomic oprtv aayi o inr aiis n lgn mgain in migration ligand and cavities inner of analysis Comparative doi:10. optr iuain f elt srcue n ratvt using reactivity and structure zeolite of simulation Computer J. Chem. Phys. Chem. J. J. Am. Chem. Soc. Chem. Am. J. Biochim. Biophys. Acta Biophys. Biochim. et al. Biophys. J. Biophys.

89 Substrate diffusion and oxidation in GMC oxidoreductases: 1021/jp502509 J. Comput. Chem. Comput. J. , 52–57 (1985). 52–57 , Faraday Discuss. Faraday J. Mol. Graph. Mol. J.

102

97 ici. ipy. Acta Biophys. Biochim. , 887–896 (2012). 887–896 , , 2571–2577 (1992). 2571–2577 ,

106 Proteins a . hs Chem. Phys. J. ) (13 May 2014). May (13 ) , 765–784 (1984). 765–784 ,

1834

14 106

Phys. Rev. B Condens. Matter Rev.Condens. Phys. B , 294–301 (1983). 294–301 , , 136–141 (1996). 136–141 , 65 , 79–92 (1997). 79–92 , , 1739–1743 (2013). 1739–1743 , ipy. J. Biophys. , 712–725 (2006). 712–725 , doi:

Proc. Natl. Acad. Sci. Acad. Natl. Proc.

. opt Chem. Comput. J. 100 10.1038/nsmb.2932 1834

92 10580–10594 , Math. Comput. Math. , 3817–3829 3817–3829 , 1957–1967 , hs Chem. Phys. Proteins Biochem. J. Mol. J.

37 55

7 , , ,