Key Players in I-DmoI Endonuclease Catalysis Revealed from

Subscriber access provided by FLORIDA ATLANTIC UNIV

Article

Key players of I-DmoI endonuclease catalysis revealed from structure and dynamics Rafael Molina, Neva Besker, Maria Jose Marcaida, Guillermo Montoya, Jesus Prieto, and Marco D'Abramo ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00730 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on February 26, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Key players of I-DmoI endonuclease catalysis revealed from structure and dynamics Rafael Molina1, Neva Besker2, Maria Jose Marcaida1, Guillermo Montoya1,3, Jesús Prieto1, and Marco D’Abramo4,*. 1

Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Macromolecular Crystallography Group, c/Melchor Fdez. Almagro 3, 28029 Madrid, Spain. 2 CINECA, SuperComputing Applications and Innovations, via dei Tizii 6, Rome, Italy. 3 Macromolecular Crystallography Group, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. 4 Department of Chemistry, University of Rome “La Sapienza”, p.le A. Moro, 5, 00185, Rome, Italy. * To whom correspondence should be addressed Email: [email protected] KEYWORDS. Gene targeting, genetics, protein-DNA interaction, homing endonucleases, X-ray crystallography, molecular dynamics ABSTRACT: Homing endonucleases, such as I-DmoI, specifically recognize and cleave long DNA target sequences (~ 20 bp) and are potentially powerful tools for genome manipulation. However, inefficient and off-target DNA cleavage seriously limit specific editing in complex genomes. One approach to overcome these limitations is to unambiguously identify the key structural players involved in catalysis. Here we report the E117A I-DmoI mutant crystal structure at 2.2 Å resolution that, together with the wt and Q42A/K120M constructs, are combined with computational approaches to shed light on protein cleavage activities. The cleavage mechanism was related to both key structural effects, such as the position of water molecule and ions participating in the cleavage reaction, and dynamical effects due to the protein behavior. In particular, we found that when the ions and water molecules are correctly positioned for nucleophilic attack initiating the cleavage reaction, the protein perturbation pattern significantly changes between cleaved and not-cleaved DNA strands, in line with experimental enzymatic activity. The proposed approach paves the way for an effective, general and reliable procedure to analyze the enzymatic activity of the endonucleases from a very limited dataset, i.e. structure and dynamics.

INTRODUCTION Homing endonucleases (HEs) are DNA nucleases able to selectively recognize and cleave long DNA targets (12-45 bp) generating double-strand breaks (DSBs). These DSBs are processed in a conservative way by homologous recombination (HR) or by non-homologous end joining (NHEJ) that might results in insertions or deletions leading to genomic alterations (1). DNA nickases producing single strand break reduce the toxicity associated to the error-prone NHEJ repair pathway. Because of their long target DNA sequences recognized, HEs are powerful tools for genome manipulation in mammalian and plants cells (2-4). Recent studies have proved that a single strand nick rather than a DSB stimulate HR (5,6). Nickases are found in nature but can also be artificially designed by the engineering of zinc finger nucleases (ZFNs), Transcription activator-like effector (TALE) nucleases or HEs. The three-dimensional structures of several HEs indicate that these proteins adopt a similar active conformation. Spatially distinct regions within the protein domains are responsible for DNA binding and double-strand cleavage. Once the DNA is accommodated inside the concave surface formed by the antiparallel β-sheets that are present in both domains and the metal ions are captured by the acidic residues of the catalytic motifs, two active sites are formed that can proceed to cut one DNA strand each. All HEs catalyse the cleavage of dou-

ble-stranded DNA by metal-assisted hydrolytic attack leaving 4-nt 3-OH overhangs. As a general rule for nucleases, an acidbase catalysis controls the cleavage of the phosphodiester bonds. Thus, a general base activates the nucleophile by deprotonation and a general acid facilitates product formation by protonating the leaving group. Because of their exceptionally high DNA cleavage specificities, the possibility to tune the DNA target specificity of HEs represents an intense area of research. The structural characterization of several members of the HEs family has opened the quest for methods able to produce engineered proteins with cleavage activity against specific DNA targets. For example, mutation of specific residues that interact with DNA in three members of LAGLIDADG family (I-CreI, PI-SceI and I-SceI) (7-10), showed cleavage of novel targets and/or alteration of binding activity. Although high-throughput screen of a larger number of mutant/target combinations is now possible (11,12), the high number of residues possibly implicated in protein activity makes such a brute-force approach unrealistic (13). It is therefore highly desirable that computational approaches help reducing the combinatorial of mutant screens by precisely identifying key-factors involved in DNA cleavage. By combining biochemical activity and structural data of HEs, a database that identifies DNA sequences targeted by various LHEs was recently built (14).

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 10

RESULTS Structural relationship between active sites in I-DmoI wt, Q42A/K120M and E117A. The crystal structure of IDmoI in complex with its DNA target showed that residues D21 and E117 in the active site are involved in the coordination of divalent ions, which play an essential role in the catalysis (15). We have recently reported all catalytic steps followed by I-DmoI and identified three ions located at positions A, B and C in the active site, as being responsible for a “two-metal ion” cleavage mechanism (26). As a consequence of these studies and attempting to tailor I-DmoI as a nicking enzyme, we developed a new variant, I-DmoI Q42A/K120M, able to preferentially generate nicked DNA in the coding strand (27). The overall 3D structure of the mutant and wild type (wt) were very similar (Fig. 1a), and no differences were observed at site B (the active site where the coding-strand cleavage occurs). On the other hand, at site A we found that the K120M/Q42A mutant is able to completely inhibits the cleavage of the noncoding strand (NCS), although the corresponding nucleophilic water as well as the ions are correctly positioned (Fig. 1b).

However, despite these efforts, rationalizing the structural and dynamical factors affecting protein activity remains a considerable challenge. Here, we have combined experimental and theoretical approaches to I-DmoI and two of its mutants and present a possible explanation of the subtle mechanisms causing the observed different cleavage activities. In particular, analysing the perturbations due to the solvent and protein as well as water structural rearrangements, we were able to suggest a link between in silico dynamics and the experimental protein activity. MATERIALS AND METHODS Protein expression, purification, protein-DNA complex formation and crystallization. I-DmoI wild type and mutant variants were cloned, expressed and purified following earlier protocols (15,16). Protein-DNA complexes were obtained as described in Redondo et al. (17) and their crystallization conditions were similar to the wild type I-DmoI–DNA complex crystal structure, ranging from 5-6 % PEG4000, 0.07 M NaAc pH = 4.6-5.5 and 30% Glycerol. The DNA targets were purchased from Proligo and consisted of the following duplexes: 1) 5’-GCCTTGCCGGGTAAGTTCCGGCGCG-3’ and 5’CGCGCCGGAACTTACCCGGCAAGGC-3’. Individual strands in TE buffer in the presence of 50 mM NaCl were combined in a 1:1 ratio and annealed by incubating at 95°C for 10 minutes and slowly cooling to room temperature to form DNA duplexes. All of them form a 25-bp blunt-end duplex after incubation. Sample concentrations were quantified by UV spectroscopy, using the calculated extinction coefficients. Protein concentrations were further confirmed by Bradford assay. Data collection, structure solution, model building and refinement. All data were collected at 100 K, using synchrotron radiation at beamlines XALOC (ALBA, Barcelona, Spain) and at the PXI beamline (SLS, Villigen, Switzerland). Diffraction data were recorded on PILATUS 6M detectors. Processing and scaling were accomplished with XDS (18) and SCALA (19) software packages. Statistics for the crystallographic data and structure solution are summarized in Table I. The structures were solved by molecular replacement as implemented in the program PHASER (20). The search models were based on the PDB entries 2VS7 (I-DmoI-DNA-Ca2+) and 2VS8 (I-DmoI-DNA-Mn2+). The structures were then subjected to iterative cycles of model building and refinement with Coot (21) and PHENIX (22). Molecular Dynamics Simulations. The molecular dynamics simulations of the I-DmoI wild type, I-Dmol Q42A/K120M and I-DmoI E117A mutants bound to DNA were performed starting from the corresponding crystallographic structures. The version 4 of the molecular dynamics program GROMACS (23) with Amber99sb-ildn* force field (24) were used. After minimization and thermalization procedure (see Suppl. Inf.), MD simulations of 200 ns were performed, using a time step of 2 fs. The temperature was kept constant at 343 K using the velocity rescaling algorithm (25). Every 2 ps, the electric field given by the atomic charge distribution of the all atoms of the protein, the water molecules and the ions was calculated and projected on the phosphorousoxygen bond of the coding and non-coding strands. Further details on the simulation protocol and analysis are reported in the Suppl. Inf.

Figure 1. Structural comparison between I-DmoI wild-type, IDmoI Q42A/K120M and I-DmoI E117A. (a) Overall structure of I-DmoI wild-type (top) and I-DmoI Q42A/K120M (bottom) in complex with its DNA target and in the presence of catalytic ions. The sequence of the DNA target is shown at right. (b) Zoom at the active site superimposition between I-DmoI wild-type and I-DmoI Q42A/K120M in the presence of 2 mM Mn2+ and its target DNA. (c) Overall structure (left) and detailed view (right) of the I-DmoI E117A active site in the presence of 2 mM Mn2+. Fo-Fc omit map (grey) around the phosphodiester bonds cleavable is superimposed onto their corresponding refined structures. The omit maps density is contoured at 7σ. Anomalous map (red), revealing the number and position of ions, show density contoured at 5σ.

Therefore, in our quest for a nicking enzyme we were able to obtain site A with decreased activity that did not affect the cleavage at site B but were unable to tailor a nicking enzyme that cleaved preferentially at site A. Likely, this was because,

2 ACS Paragon Plus Environment

Page 3 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology could not be cleaved, the Mn2+ and the water in the active site A as well as the residues responsible for coordination were in the same position as in the wild type. The main differences in active site A were limited to the rearrangement of the non-coding strand mentioned above, which caused a rotation in the scissile phosphate of the non-coding strand (Fig. 2b-c). However, it still remained unclear how such an effect completely inhibited the cleavage reaction at site A. Moreover, the changes in cleavage activity of wt vs. Q42A/K120M highlighted that even for almost indistinguishable structures of the protein-DNA complex, with metal ions located in the right positions, the double mutation induced a nickase activity (i.e. only the coding-strand was cleaved). As protein biological functions are chiefly regulated by their structural dynamics, the above results strongly suggested that a more detailed understanding of the enzymatic activity as well as its dependency on the active site environment should be addressed by a dynamical description of the system. Therefore, starting from the I-DmoI wt, E117A and Q42A/K120M mutants, we studied the protein-DNA complexes dynamical behavior by means of all-atoms molecular dynamics (MD) simulations to try and rationalize the cleavage activity observed experimentally. Global dynamics of I-DmoI wt, Q42A/K120M and E117A. We first compared the global dynamical properties of the proteins as sampled by MD trajectories. The root mean square deviations (rmsd) from the starting structure remain below 2 Angstrom for all the simulated systems (Fig. S1), indicating that no conformational changes occurred and that mutations did not affect such a property. The residue fluctuations of the Q42A/K120M were very similar (albeit slightly enhanced) to those of the wt protein (Fig. S2). A detailed analysis showed that the most fluctuating regions were prevalently located in the turns connecting the β-sheets and between β-sheets and helical structures. This was in line with the high conservation of the protein secondary structures observed all along the trajectories. In the case of the single mutant E117A, the fluctuations were significantly enhanced with respect to both wt and Q42A/K120M proteins, especially in two regions located at the protein-DNA interface (i.e. between the residues 126-133 and 148-155). To check whether mutations affect the global motions of the proteins, we applied principal components analysis - a procedure based on the diagonalization of the covariance matrix built from the atomic fluctuations - to extract the principal motion directions (eigenvectors) of the protein (i.e. where the most relevant part of the protein motions is concentrated). Using this approach - called Essential Dynamics (ED) Analysis (28) - we found that the first ten eigenvectors (associated to the highest eigenvalues) of each trajectories expressed, at least, 50% of the whole protein motions in all the simulated systems. Noteworthy, analysis of the first three eigenvectors showed that, the principal motions of wt and in the E117A mutant were not concentrated in specific regions, but were well distributed all along the protein main chain (Fig. S3). On the other hand, in the double-mutant Q42A/K120M we observed a relevant contribution of six residues (between S67 and V72 and far from the mutated residues) to the first 3 eigenvectors. Such a region corresponded to the most fluctuating region of the protein. However, the similarities between the first ten eigenvectors for the three systems, as calculated by the root-mean-square-inner product, were sensibly high

altering site B resulted in the inhibition of the cleavage reaction (27). To shed some light on the role played by one active site on the other in the cleavage reaction, the E117A and D21A mutants were expressed. The D21A variant failed to crystallize even though the protein was properly folded and bound to the 25 bp double stranded DNA in a similar way as the I-DmoI wild type (wt). On the other hand, the E117A mutant bound to a 25 bp double-stranded DNA formed crystals that diffracted to 2.2 Å and the structure was solved in complex with Mn2+ ions (data were collected at the Mn2+ anomalous peak to confirm the presence of the Mn2+ ions). As expected, the DNA was not cleaved and the protein backbone did not substantially change with respect to the structure of wt bound to the substrate (C-alpha rmsd 0.32Å, Fig. 1c). However, as revealed by the anomalous signal corresponding to the Mn2+ ions, while at site A their positions were equivalent, they differed at site B (Fig. 1c, 2a). Furthermore, as electron density maps showed, DNA conformation at the active centre of IDmoI E117A – DNA complex was also altered compared to the wild type complex structure (Fig. 1c). To avoid biases, the comparison was performed using equivalent non-cleaved states of wild type and E117A complex structures. This revealed changes in the conformation of four nucleotides (2TstrandB, 1TstrandB, -1AstrandB, -2CstrandB) in the non-coding strand that disturbed base pairing for -1AstrandB (Fig. 2a).

Figure 2. I-DmoI E117A structure comparison analysis. (a) Overall structure superimposition between DNA from I-DmoI E117ADNA complex (orange) and DNA from I-DmoI wt-DNA complex at ground state (blue) active centers. (b) Zoom at the active site superimposition between I-DmoI wild-type and I-DmoI E117A. (c) Conformational changes at the active site B as a consequence of metal moiety at this position between I-DmoI wild-type and IDmoI E117A .

This distortion was likely due to the shift of the Mn2+ ion in the active site B in the absence of the side chain of E117 (Fig. 2b). Active site B was now empty and the Mn2+ ion had moved 5.4 Å away from its original position being now coordinated by O1PstrandA from 3GstrandA, O1PstrandB from -2CstrandB and 4 water molecules (Fig. 2c). The phosphates that coordinated it were the same as in the wt structure, but the phosphate from -2CstrandB was displaced by 5.2 Å (Fig. 2c). Although the lack of ion in active site B explained why the coding strand



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(RMSIP > 0.65 for both the E117A and Q42A/K120M versus the wt), thus indicating that mutations did not introduce relevant differences in the protein principal motion directions. The structural analysis of the extremes of the projections on the essential subspace as well as trajectory projections on the essential planes as defined by the first two eigenvectors confirmed that no relevant conformational changes occur along the simulations in the three systems (see Fig. S4). These results made clear that the global protein motions did not explain the distinct enzymatic activities observed experimentally (discussed in the Experimental results section), suggesting that an in-depth local analysis of the effect of protein dynamics on the cleavage site was required. Local dynamics of I-DmoI wt, Q42A/K120M and E117A. It is well known that the cleavage of the P-O bond depends on structural rearrangements of the metal ions and water molecules around the cleavage site (29,30). The analysis of the structural and dynamical properties of the systems in those sites as obtained by means of molecular dynamics simulations suggested that the wt and the Q42A/K120M protein had very similar behaviour: in both systems, the two metal ions remained near the initial position, between the LAGLIDAG protein motif and the backbone of the nucleotides 3GstrandA and -2CstrandB as found in the crystal structures (Fig. S5). On the other hand, the structural rearrangement observed in the E117A crystal structure described in the previous section, affected the network of the water molecules around the cleavage site B, enhancing the number of solvent molecules coordinating the ions and their residence time (Fig. 3).

Page 4 of 10

of the electric field due to the charge distribution of the environment (i.e. protein, DNA, solvent and ions) and then projecting it onto those bonds. The use of the electric field as the observable to measure the effect of the protein and, in general, of the environment on the reaction centre has been successfully employed to study a series of chemical-physical processes involving electronic rearrangements, where the environment significantly modified the process behaviour (31-35). The distributions of the projected electric field (hereafter, refereed to as EfProj) for both the DNA strands in the three systems are shown in Fig. 4.

Figure 4. Projection of the electric field (obtained from the atomic charge distribution along the trajectory) on the P-O bond for the wt and Q42A/K120M proteins (top panel) and for the wt and E117A proteins (bottom panel). CS and NCS refer to the Coding Strand and Not-Coding Strand, respectively.

Remarkably, the EfProj distributions for the coding strands were quite similar for wt and Q42A/K120M, the latter being only slightly left-shifted and broader (Fig. 4, top panel). Instead, the EfProj distributions for the coding strands - which were cleaved only by the wt – were significantly different, highlighting a change in the sign of the perturbation. The EfProj distributions of the E117A (Fig. 4, bottom panel) suggested that the cleavage inhibition in the non-coding strand was due to the change in the perturbation due to the environment, similar to what happens in the NCS of the Q42A/K120M, previously described. In the E117A case, the shift of the EfProj distribution might be due in principle to both the rotation of the NCS scissile phosphate and a different environmental perturbation induced by the mutation. However, what we measured here is the final result of the combination of these two effects, and the proposed observable – the EfProj - correlates well with the observed protein enzymatic activity. Interestingly, the EfProj distribution for the CS in the E117A was almost identical to the same strand in the wt protein: in this case, the absence of the cleavage is probably due to the lack of the metal ion in the site B, in line with our previous findings (26). The corollary of such a similar behaviour observed in the E117A and wt coding-strands is that the ion, although highly charged and located near the active site, is not the main key-player of the perturbation, which is indeed due to the whole environment (i.e. all the protein atoms, water and ions). To further understand the protein regions responsible for the change in the perturbation, we performed an atomic-based

Figure 3. Distribution of the water molecule number at a distance < 0.6 nm from the Mn ion (left panel) and their residence time (right panel) as provided by MD simulations.

These data confirmed that, in the case of E117A, the cleavage of the Coding-Strand (CS) A was inhibited because of the reorganization of the ion and water near the site B which produced unfavourable configurations for the P-O bond hydrolysis of the coding strand A (29,30). In the case of the cleavage sites A and B of wt and Q42A/K120M mutant as well as the cleavage site A of E117A, the trajectories showed a very similar behaviour for solvent and structural properties around the cleavage sites. The water molecule are correctly positioned around the Mn2+ ions and several configurations compatible with the first step of the phosphodiester hydrolysis - starting when one water molecule, acting as nucleophile, attacks the P-O bond (26) - were sampled along the trajectories (see Fig. S5 for wt case). The difference in enzymatic activity between the wt and Q42A/K120M - where only the latter acted as a nickase – as well as the complete absence of cleavage in the E117A, indicated that a different factor influencing the cleavage reaction should exist. Our previous work in the theoretical modelling of chemical reactions in a complex environment (31-33) made possible to analyse the perturbation on the P-O bonds in terms


Page 5 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


analysis for the coding and not-coding strand in the wt and Q42A/K120M proteins. The first essential finding of such an analysis was that the major contribution to the perturbation was mainly due to few and limited regions distributed all along the protein structure (Fig. 5 and Table I). For example, we found that, in the wt, relevant contributions to the perturbation were due to the regions corresponding to the α1, β2, α4, β5 and β6 (following the secondary structure scheme reported in Fig. 6). To gain insight in different perturbation pattern, it was of primary interest to compare the residue-based perturbation contribution between the wt and Q42A/K120M for both the strands. In the CS case, which was cleaved by both the proteins, the analysis shows a very similar behaviour with a limited difference in the region between residue 113 and 117. On the other hand, large differences were found when the wt and Q42A/K120M are compared in the NCS case (Fig. 5, red lines). Here, it emerged that the α1 and β2 regions of the Q42A/K120M provided a contribution of opposite sign with respect to the same strand in the wt.

Figure 6. Sequence and secondary structure of the wt I-DmoI as obtained from the crystallographic structure.

residue 17 - a spatially close zone located in the proteinDNA interface - which greatly contributes to the perturbation profile. Such behaviour, together with the EfProj distributions (Fig. 5), indicates that the Q42A/K120M, which presents no significant change in the global conformational behaviour, is still able to affect the protein dynamics at local level. This, in turn, modifies the perturbation of the protein on the enzymatic reaction centre, i.e. the P-O bond. DISCUSSION We combined experimental and computational data of the wt I-DmoI and two mutants to try to find a rationale behind their different enzymatic activity. To this end, we propose to analyse the protein dynamical behaviour as obtained by molecular dynamics simulations in terms of active site structure and environmental effect on the scissile P-O bond as measured by the electric field exerted by the environment on that bond. The use of the latter was proposed to account for subtle differences between the three systems which are missing when the protein-DNA dynamical behaviours were analysed and compared at global level. Our data indicate that the cleavage reaction occurs –when both the cations are correctly positioned in the active site and the environment exterts a specific perturbation pattern on the P-O bond.The wt I-DmoI protein - able to cleave both DNA strands - was taken as reference to characterize two of its mutants (e.g. in the case of wt, both the conditions are satisfied). In the Q42A/K120M, the correct positioning of the ions is satisfied in both the active sites, but for the non-coding strand – experimentally found not cleaved - the environment provides a different perturbation behaviour on the P-O bond. The different perturbation pattern with respect to the wt is also observed in the non-coding strand of the E117A mutant, correlating well with the experimentally observed absence of cleavage in this DNA strand. Finally, in the coding strand of the E117A, which is found not cleaved, the perturbation is compatible with the P-O cleavage but the lack of the ion does not.

Figure 5. Residue based contributions to the projection of the electric field on the P-O bond for (a) the wt (top panel) and for the Q42A/K120M (bottom panel). Black lines: Coding Strand; red lines: Not-Coding Strand. (b) Cartoon representation of the wt protein. The colours of the residues express their contribution to the perturbation on the P-O bond of the Coding-Strand. In addition, the contribution of the α4 region showed 4 peaks in contrast to the wt where a single positive peak was observed. From this analysis we deduce that the mutations change the perturbation behaviour only on the NC strand, leaving almost unaffected that of the CS strand with respect to the wt. In addition, such an effect is propagated up to the region around

CONCLUSIONS



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

We have analysed the I-DmoI enzymatic activity in terms of simple structural and dynamical observable, comparing experimental X-ray structures, cleavage essays and molecular dynamics simulations. Although a quantitative modelling of enzymatic analysis does require the characterization of the enzymatic effect on the transition state of the chemical reaction, our results show that cleavage of DNA by I-DmoI endonucleases occurs only when the chemical species required for the cleavage (i.e. nucleophilic water and metal ions) are in a well defined position and the environment provides a specific environmental perturbation on the P-O scissile bond. The use of these observables, which might easily evaluated by structural and dynamical analysis of the protein-DNA complex, provides a more detailed understanding of the effect of the mutations in the enzymatic behavior, facilitating the design of synthetic endonucleases with tailored specificities.

No. Molecules in a.u.

PDB code

Q42AK120M DNA WT Mn2+

2VS8

5A0W

5AK9

P21

P21

P21

107.13, 70.63, 106.96

106.93, 69.77, 106.98

107.53, 70.88, 107.68

β (°)

119.85

120.06

119.78

Wavelength

1.00

1.5490

0.8726

Resolution (Å)

45.63-2.10 (2.15-2.10)*

29.22-2.20 (2.32-2.20)*

42.95-2.59 (2.73-2.59)*

Rmerge

0.05 (0.45)

0.06 (0.32)

0.10 (0.78)

Mean I / sI

12.8 (2.3)

16.7 (2.8)

10.8 (1.9)

Completeness (%)

99.7 (99.5)

96.9 (95.1)

97.5 (92)

3.0 (2.8)

3.1 (3.0)

3.2 (3.1)

25.76-2.10

29.22-2.20

42.94-2.60

Redundancy

Both strands cleaved

None

Coding strand cleaved

6

6

6

Protein

4573

4618

4610

DNA

3063

3057

3060

Ions

6

18

14

392

401

233

Bond lengths (Å)

0.012

0.007

0.007

Bond angles (°)

1.698

1.237

1.285

Water R.m.s. deviations

*Values in parentheses are for highest-resolution shell. One crystal was used to solve each structure.

Table 2. List of the residues primarily contributing to the perturbation of the protein on the cleavage site (high and medium refer to values of the electric field projection above 0.01 a.u. and between 0.05 and 0.01, respectively; when the electric field projection is negative a minus sign is present before the residue number).

Cell dimensions a, b, c (Å)

3

No. Atoms

Data collection Space group

3

No. Ions at a.s.

Table 1. Data collection and refinement statistics. E117A DNA WT Mn2+

3

DNA strands cleaved?

TABLES.

WT DNA WT Mn2+

Page 6 of 10

High

Medium

WT CS

17; 126

-39; 113; -116

WT NCS

116

17; 39; -113; 151; -154

Q42A/K120M CS

17

-39; -113; 126

Q42A/K120M NCS

ASSOCIATED CONTENT Supporting Information. Supplementary figures of computational analysis. The crystallographic coordinates were deposited in the Protein Data Bank (PDB codes: 2VS8, 5A0W and 5AK9). This material is available free of charge via the Internet at http://pubs.acs.org.

Refinement Resolution (Å) No. Reflections Rwork / Rfree

76811

120107

81107

0.20/0.25

0.17/0.21

0.17/0.22

17; -39; 113; -115; 116; 120

AUTHOR INFORMATION Corresponding Author * [email protected].


Page 7 of 10

ACS Chemical Biology 13. Paques, F. and Duchateau, P. (2007). Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Current gene therapy, 7, 49-66. 14. Taylor, G.K., Petrucci, L.H., Lambert, A.R., Baxter, S.K., Jarjour, J. and Stoddard, B.L. (2012). LAHEDES: the LAGLIDADG homing endonuclease database and engineering server. Nucleic acids res., 40, W110-116. 15. Marcaida, M.J., Prieto, J., Redondo, P., Nadra, A.D., Alibes, A., Serrano, L., Grizot, S., Duchateau, P., Paques, F., Blanco, F.J., and Montoya, G. (2008). Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering. P. Natl Acad Sci USA, 105, 16888-16893. 16. Prieto, J., Redondo, P., Padro, D., Arnould, S., Epinat, J.C., Paques, F., Blanco, F.J. and Montoya, G. (2007). The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage. Nucleic acids res., 35, 3262-3271. 17. Redondo, P., Prieto, J., Ramos, E., Blanco, F.J. and Montoya, G. (2007). Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-Dmo-I in complex with its target DNA. Acta crystallogr. F, 63, 1017-1020. 18. Kabsch, W. (2010) XDS. Acta crystallogr. F, 66, 125-132. 19. Evans, P. (2006). Evans, P. (2006). Acta crystallogr. D, 62, 72-82. Acta crystallogr. D, 62, 72-82. 20. McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C. and Read, R.J. (2007). Phaser crystallographic software. J. Appl. Crystallogr., 40, 658-674. 21. Emsley, P., Lohkamp, B., Scott, W.G. and Cowtan, K. (2010). Features and development of Coot. Acta crystallogr. D, 66, 486-501. 22. Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., and Zwart, P.H. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallogr. D, 66, 213221. 23. Hess, B., Kutzner, C., van der Spoel, D. and Lindahl, E. (2008). GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theo. Comp., 4, 435-447. 24. Hornak, V., Abel, R., Okur, A., Strockbine, B., Roitberg, A. and Simmerling, C. (2006). Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins, 65, 712-725. 25. Bussi, G., Donadio, D. and Parrinello, M. (2007). Canonical sampling through velocity rescaling. J. Chem. Phys., 126, 014101. 26. Molina, R., Stella, S., Redondo, P., Gomez, H., Marcaida, M.J., Orozco, M., Prieto, J. and Montoya, G. (2015). Visualizing phosphodiester-bond hydrolysis by an endonuclease. Nat. Struct. Mol. Biol., 22, 65-72. 27. Molina, M., Marcaida. M.J., Redondo, P., Marenchino, M., Duchateau, P., D'Abramo, M., Montoya, G. Prieto, J. (2015). Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification. J. Biol. Chem., 290(48):28727-36. 28. Amadei, A., Linssen, A.B. and Berendsen, H.J. (1993). Essential Dynamcis of Proteins. Proteins, 17, 412-425. 29. Dupureur, C.M. (2010). One is enough: insights into the two-metal ion nuclease mechanism from global analysis and computational studies. Metallomics, 2, 609-620. 30. Galburt, E.A. and Stoddard, B.L. (2002). Catalytic mechanisms of restriction and homing endonucleases. Biochemistry, 41, 13851-13860. 31. Amadei, A., D'Abramo, M., Daidone, I., D'Alessandro, M., Di Nola, A., Aschi, M. (2007). Statistical mechanical modelling of chemical reactions in complex systems: the kinetics of the haem carbon monoxide binding-unbinding reaction in myoglobin. Theor. Chem. Acc. , 117, 11. 32. Aschi, M., D'Abramo, M., Ramondo, F., Daidone, I., D'Alessandro, M, Di Nola, A., Amadei, A. (2006). Theoretical modeling of chemical reactions in complex environments: the intramolecular proton transfer in aqueous malonaldehyde. J. Phys. Org. Chem, 19, 13.

Funding Sources

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). This work was supported by the Ministero dell'Istruzione, Università e Ricerca [R.Levi-Montalcini fellowship to M.D.], Ministerio de Economía y Competitividad [JCI-2011-09308 to R.M.]. The Center for Protein research is supported by the Novo Nordisk Foundation grant NNF14CC0001.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the Swiss Light Source and XALOC beamline staff from the Spanish Synchrotron Radiation Facility for their support. We acknowledge the CINECA award under the ISCRA initiative and the Dept. of Chemistry for the availability of high performance computing resources.

REFERENCES 1. Mimitou, E.P. and Symington, L.S. (2009). Nucleases and helicases take center stage in homologous recombination. Trends Biochem Sci, 34, 264-272. 2. Choulika, A., Perrin, A., Dujon, B. and Nicolas, J.F. (1995). Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol. Cel.. Biol., 15, 1968-1973. 3. Rouet, P., Smih, F. and Jasin, M. (1994). Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. P. Natl Acad Sci USA, 91, 6064-6068. 4. Rouet, P., Smih, F. and Jasin, M. (1994). Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cel.. Biol., 14, 8096-8106. 5. Metzger, M.J., McConnell-Smith, A., Stoddard, B.L. and Miller, A.D. (2011). Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template. Nucleic acids res., 39, 926-935. 6. van Nierop, G.P., de Vries, A.A., Holkers, M., Vrijsen, K.R. and Goncalves, M.A. (2009). Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease. Nucleic acids res., 37, 5725-5736. 7. Doyon, J.B., Pattanayak, V., Meyer, C.B. and Liu, D.R. (2006). Directed evolution and substrate specificity profile of homing endonuclease I-SceI. J. Am. Chem. Soc., 128, 2477-2484. 8. Gimble, F.S., Moure, C.M. and Posey, K.L. (2003). Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system. J. Mol. Biol., 334, 993-1008. 9. Seligman, L.M., Stephens, K.M., Savage, J.H. and Monnat, R.J., Jr. (1997). Genetic analysis of the Chlamydomonas reinhardtii ICreI mobile intron homing system in Escherichia coli. Genetics, 147, 1653-1664. 10. Sussman, D., Chadsey, M., Fauce, S., Engel, A., Bruett, A., Monnat, R., Jr., Stoddard, B.L. and Seligman, L.M. (2004). Isolation and characterization of new homing endonuclease specificities at individual target site positions. J. Mol. Biol., 342, 31-41. 11. Arnould, S., Chames, P., Perez, C., Lacroix, E., Duclert, A., Epinat, J.C., Stricher, F., Petit, A.S., Patin, A., Guillier, S., Rolland S., Prieto J., Blanco F.J., Bravo J., Montoya G., Serrano L., Duchateau P. and Pâques F.(2006). Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. J Mol. Biol., 355, 443-458. 12. Chames, P., Epinat, J.C., Guillier, S., Patin, A., Lacroix, E. and Paques, F. (2005). In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination. Nucleic acids res., 33, e178.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

33. Aschi, M., D'Alessandro, M., Pellegrino, M., Di Nola, A., D'Abramo, M., Amadei, A. (2008). Intramolecular charge transfer in π-conjugated oligomers: a theoretical study on the effect of temperature and oxidation state. Theor. Chem. Acc., 119, 8. 34. D'Abramo, M., Orozco, M. and Amadei, A. (2011). Effects of local electric fields on the redox free energy of single stranded DNA. Chem. Comm., 47, 2646-2648. 35. D'Alessandro, M., Aschi, M., Paci, M, Di Nola, A., Amadei, A. (2004). Theoretical modeling of enzyme reactions: the electron transfer of the reduction step in CuZn Superoxide Dismutase. J. Phys. Chem. B, 108, 5.


Page 8 of 10

Page 9 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SYNOPSIS TOC. The structural and dynamical behavior of the I-DmoI endonuclease bound to the DNA, studied by means of a combined experimental and theoretical approach, highlights that cleavage of DNA occurs only when both the chemical species required for the reaction (i.e. nucleophilic water and metal ions) are in a well defined position and the environment provides a specific environmental perturbation on the P-O scissile bond.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

SYNOPSIS TOC. The structural and dynamical behavior of the I-DmoI endonuclease bound to the DNA, studied by means of a combined experimental and theoretical approach, highlights that cleavage of DNA occurs only when both the chemical species required for the reaction (i.e. nucleophilic water and metal ions) are in a well defined position and the environment provides a specific environmental perturbation on the P-O scissile bond. 860x613mm (72 x 72 DPI)

ACS Paragon Plus Environment

Page 10 of 10

Key Players in I-DmoI Endonuclease Catalysis Revealed from

Recommend Documents