Role of Magnesium Ions in DNA Recognition by the Ecorv Restriction Endonuclease ⇑ Mai Zahran A, Tomasz Berezniak A, Petra Imhof A, , Jeremy C

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FEBS Letters 585 (2011) 2739–2743

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Role of magnesium ions in DNA recognition by the EcoRV restriction endonuclease ⇑ Mai Zahran a, Tomasz Berezniak a, Petra Imhof a, , Jeremy C. Smith b

a Computational Molecular Biophysics, IWR, University of Heidelberg, Heidelberg, Germany b University of Tennessee, Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

article info abstract

Article history: 2þ The restriction endonuclease EcoRV binds two magnesium ions. One of these ions, MgA , binds to the Received 17 June 2011 phosphate group where the cleavage occurs and is required for catalysis, but the role of the other Revised 20 July 2011 2þ ion, MgB is debated. Here, multiple independent molecular dynamics simulations suggest that Accepted 25 July 2011 2þ MgB is crucial for achieving a tightly bound protein–DNA complex and stabilizing a conformation Available online 5 August 2011 2þ that allows cleavage. In the absence of MgB in all simulations the protein–DNA hydrogen bond net- Edited by Christian Griesinger work is signiﬁcantly disrupted and the sharp kink at the central base pair step of the DNA, which is observed in the two-metal complex, is not present. Also, the active site residues rearrange in such a way that the formation of a nucleophile, required for DNA hydrolysis, is unlikely. Keywords: Nuclease Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Restriction enzyme Protein–DNA interaction Divalent metal ions Mg2+

1. Introduction to the scissile phosphate group, and ‘‘Metal B’’ is bound to Glu45 and Asp74. In this mechanism Metal A is responsible for activating

Restriction endonucleases recognize specific DNA sequences the nucleophile by lowering its pKa, whereas Metal B reduces unfa- and hydrolyze the phosphate-diester bonds in the DNA backbone vorable electrostatic repulsion during the formation of a pentava- in the presence of divalent metal ions [1]. EcoRV, a homodimeric lent transition state and facilitates the departure of the leaving type II restriction enzyme found in Escherichia coli, recognizes spe- group (see Fig. 1). cifically the unmethylated sequence 50-GATATC-30 and cleaves it at An alternative, one-metal-ion mechanism was also proposed, in the central base pair step (TA) [2]. When bound to EcoRV, this cen- which only Metal A is essential for catalysis [15], whereas Metal B tral base pair is known experimentally to exhibit a sharp kink has an activity-regulating role [5,11,16]. Mutation of Glu45, which which corresponds to a roll angle of 50°. In contrast, the roll angle binds Metal B, to alanine leads to a still partially active enzyme of DNA in the unbound canonical B-form is 1.5° [3]. [15]. In contrast, mutations of Asp74 and Asp90, both coordinating Divalent metal ions (most commonly Mg2+) play a functional Metal A, render the mutant enzymes catalytically inactive. This role in both the catalytic process and in the formation of the speci- suggests that Asp74 and Asp90 are crucial for catalysis. A recent ficity-determining contacts between the protein and the DNA [4,5], QM/MM study of the catalytic mechanism of EcoRV showed that and possess a substantially larger affinity for the enzyme–DNA only Metal A has a direct catalytic role in the cleavage reaction complex at the cognate than at non-cognate sites [6]. In the absence by activating a water molecule that protonates the leaving group of divalent metal ions EcoRV demonstrates no catalytic activity, [16]. Recent experimental and computational studies performed although it can still bind to DNA non-specifically [7–9]. on restriction enzymes from the EcoRI subfamily (BamHI and Eco- Crystal structures of EcoRV in complex with specific DNA RI), which are structurally similar to the EcoRV enzymes, also sup- sequences revealed one or two metal ions at the active site at port the one-metal-ion mechanism [11]. Molecular dynamics (MD) various positions, indicating diverse catalytic scenarios for the simulations of BamHI suggest that Metal A binds more strongly to enzymatic cleavage reaction [10,11]. A two-metal-ion mechanism the enzyme than Metal B [17], questioning the importance of Metal was proposed on the basis of the available crystal structures in B for catalysis. which both metal ions have a direct catalytic function [12–14]. Computational work has demonstrated the usefulness of MD ‘‘Metal A’’ is bound to the active site residues Asp74, Asp90 and simulation in understanding the interplay between the structural properties of nucleic acids and their interactions with the protein ⇑ Corresponding author. partners [18–20]. Here, MD simulations are performed to examine 2þ E-mail address: [email protected] (P. Imhof). the role of Metal B ðMgB Þ in the structure of the EcoRV–DNA

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.07.036 2740 M. Zahran et al. / FEBS Letters 585 (2011) 2739–2743

Fig. 1. Positions of the two metal ions at the active site. The arrows indicate nucleophilic attack and leaving group protonation.

2þ complex. Removal of MgB results in active site rearrangement, contrast, for 1TA q drops within the first 4 ns from the initial 50° 2þ and changes the coordination partners of Metal A ðMgA Þ so as to angle to 10° ±2°. A similar roll angle of 10° ±2° is also found render nucleophile generation unlikely. All the results suggests for the non-cognate AT simulation. 2þ that, although MgB is not directly involved in the catalytic reac- The data shown in Fig. 2 demonstrate that the deletion of the 2þ tion, it does play an important role in providing a tightly-bound MgB induces a structural change in the DNA that leads to removal 2þ complex and maintaining the appropriate geometry and stability of most of the roll. The presence of MgB may thus be essential to of the catalytic pocket for cleavage. keep the DNA in the optimal conformation for cleavage. Crystal structures of EcoRV–DNA complexes determined in distinct crystal 2. Materials and methods lattices show a series of states with DNA bent to varying degrees [24], but catalytic activity in the crystalline state was observed The X-ray crystallographic coordinates of the EcoRV–DNA com- only in the most highly bent structure, which corresponds to plex (1sx8) were used as the starting structure. Here, we per- approximately q =50° at the central step TA [24]. In the light of formed five independent 60 ns simulations of EcoRV complexed this result, the lower roll angle observed in the simulations of to the cognate DNA sequence in the presence of one Mg2+ ion at 1TA suggests that DNA cleavage is not likely in the one-ion the A site (1TA). The simulations were performed with the program complex. NAMD using the CHARMM27 force field. For a detailed description of the methods see the Supplementary materials. 3.2. Comparison of the protein–DNA interactions

3. Results and discussion A schematic view of the most stable hydrogen-bond interactions formed between the two subunits of the protein and the MD simulations of cognate-DNA–EcoRV complexed with only two strands of the DNA is presented in Fig. 3 (see also Table B in 2þ the Supplementary materials). A distinction is made between MgA (1TA) are compared to results of simulations published previously of cognate-DNA–EcoRV (2TA) and non-cognate-DNA– 2þ 2þ EcoRV (AT), both complexed with two ions (MgA and MgB ) [19]. The non-cognate DNA sequence GAATTC is known not to be cleaved by EcoRV [21]. The observed changes in the active site structure were the same in each of the ﬁve independent simulations performed. Data com- paring the individual simulations are presented in the Supplemen- tary materials.

3.1. Roll angle

The roll angle (q) is a rotational helical parameter measuring the angle between the planes formed by two consecutive base pairs, and is the primary mode of DNA bending [22–24]. Fig. 2 shows the roll angle of the central base pair of the DNA as a function of simulation time for the 2TA, 1TA and AT complexes for one simulation. Data for each run are shown in Fig. A of the Supplementary materials. The roll angle is about 47° ±3° during the 2TA simulation in accordance with the roll angle observed in Fig. 2. Roll angle at the central step as a function of simulation time (ns) for the the crystal structure of the cognate EcoRV–DNA complex [2,24] cognate EcoRV–DNA complex with two Mg2+ ions, 2TA (orange), with one Mg2+ ion, and of the cognate EcoRV–DNA complex in solution [25].In 1TA (red) and the non-cognate AT complex (blue). M. Zahran et al. / FEBS Letters 585 (2011) 2739–2743 2741

Fig. 3. Schematic view of the hydrogen-bond interactions of the DNA with each subunit of the protein for the 2TA and 1TA complexes. Specific interactions are represented as blue dotted lines and interactions with the backbone are shown with dashed gray lines. The occupancies of the hydrogen bonds are highlighted by a color gradient from pale yellow for low occupancy to dark green for high occupancy. hydrogen bonds with specific atoms of the base and with atoms of hydrogen-bond pattern of 1TA is similar to that previously ob- the DNA backbone (designated as ‘‘non-specific’’). served in simulations of the non-cognate AT complex in which The hydrogen-bond patterns of 2TA and 1TA differ strongly. In DNA cleavage does not occur [19]. 1TA the hydrogen bond pattern of subunit A is perturbed by the The above findings should also be considered in the light of the 2þ deletion of MgB . Residues Thr93, Tyr138, Arg140 and Arg226, no reduced interaction observed in simulations of the complex of longer form hydrogen bonds with the DNA, and Ser112 and EcoRV with DNA methylated at the first adenine of the recognition

Ser41 form hydrogen bonds with the backbone atoms of the DNA sequence GAmTATC (mTA) [19], in which the methylation prevents but shifted by one nucleotide compared to 2TA. Furthermore, the Gly184 and Tyr95 hydrogen bonds are less occupied in 1TA than in 2TA. In contrast, subunit B does not exhibit differences as drastic as in subunit A. Nevertheless, Tyr138, Tyr95 and Arg226 still exhi- 2þ bit weaker hydrogen bond occupancies in the absence of MgB , and in 1TA Arg140 of both subunits does not hydrogen bond with either of the two strands of the DNA. 2þ The absence of MgB weakens the protein–DNA hydrogen bonds, consistent with the overall reduction in the protein–DNA interaction energy (Table A of the Supplementary materials). How- ever, only the non-specific interactions are weakened in absence of 2þ MgB , whereas the specific interactions are still very strong, as was also found for the non-specific complex AT [19]. Furthermore, the 1TA and AT complexes exhibit asymmetry in the hydrogen-bond pattern of the protein subunits, whereas the pattern of 2TA is symmetric [19]. The presence of weak hydrogen bonds and asymmetry in the hydrogen-bond pattern are both char- acteristics of non-cognate or weakly-bound protein–DNA complexes, as discussed in Refs. [19,26]. 2þ In the absence of MgB , the EcoRV–DNA complex is significantly less tightly formed than in the presence of both metal ions. The strong protein–DNA interactions absent in 1TA are required to provide sufficient energy to drive the sharp kink of the DNA at Fig. 4. Expanded view of the catalytic site groups of the 2TA. The arrows indicate 2þ the central step [19]. Moreover, the weak and asymmetric the changes that occur upon deletion of MgB . 2742 M. Zahran et al. / FEBS Letters 585 (2011) 2739–2743

2þ the formation of a crucial hydrogen bond (Asn185–Ade4) between otherwise coordinate MgB in the two-metal complex. Asp36, coor- 2þ the protein and the DNA and leads to reduced hydrogen-bond dinating MgB in the two-metal complex, changes its orientation occupancies relative to the non-methylated case. In the mTA sim- away from Glu45 and towards Arg140 with which it forms a ulations the binding of the protein to the methylated-cognate DNA hydrogen bond. Consequently, Arg140 loses its strong interaction was also not tight and no kink was found at the central base pair with the DNA, see Figs. 3 and 5. Lys38 changes from hydrogen step, similar to the behavior of the one-metal 1TA analyzed in bonding Asp36 to hydrogen bonding Glu45 (Fig. 5B). Asp74 reori- 2þ the current work. ents and double coordinates MgA , making the Glu45–Asp74 hydrogen bond longer than in 2TA. 3.3. Comparison of the active site pocket Comparison of the hydrogen-bond occupancies in the active sites of the two complexes shows that four of the hydrogen bonds 2þ Upon removal of MgB , the active site structure of 1TA differs that are formed in 2TA are not formed in 1TA (indicated by an from 2TA. The distance changes between the residues within the occupancy of zero in Fig. 5D). However, eight new hydrogen bonds catalytic pocket, are shown in Fig. 4 and listed in Table D of the are formed in 1TA that are absent in 2TA (compare Fig. 5C and D). 2þ Supplementary materials. Representative snapshots of the cata- The deletion of MgB is also likely to directly impact catalysis. In 2þ lytic pockets and a schematic view of the interactions within the 1TA Asp90 coordinates MgA with only one carboxyl oxygen atom, active sites of 2TA and 1TA are shown in Fig. 5A and B. A complete the second carboxyl oxygen atom forming a very stable hydrogen list of hydrogen bonds for both subunits is given in Table C of the bond with Lys92, as can be seen also by the shortened Asp90– Supplementary materials. Lys92 distance (Fig. 5B). Asp90 has been found to play an impor- 2þ 2þ In the presence of MgB , Glu45 chelates MgB , Asp74 bridges the tant role in catalysis by acting as a general base. A computational 2þ two metal ions and Asp90 double coordinates MgA (cf. Fig. 5A). analysis of the hydrolysis reaction of the phosphodiester backbone 2þ The absence of MgB leads to a reorganization of the residues that of DNA in EcoRV indicated that proton transfer from the attacking

Fig. 5. (A and B) Expanded view of the catalytic site groups from the MD simulation of 2TA and 1TA complexes, respectively. The hydrogen bonds are represented as thick dashed lines and the metal ion coordinations as thin dotted lines. (C and D) Schematic view of the hydrogen-bond and metal ion interactions at the catalytic sites of 2TA and 1TA, respectively. The occupancies of the hydrogen bonds are highlighted by a color gradient from pale green for low occupancy to dark green for high occupancy. The thin dotted lines refer to the interactions with the metal ions, in orange for 2TA and in red for 1TA complexes. M. Zahran et al. / FEBS Letters 585 (2011) 2739–2743 2743 water molecule to Asp90 generates the nucleophile [16]. However, [6] Thielking, V., Selent, U., Khler, E., Landgraf, A., Wolfes, H., Alves, J. and Pingoud, 2+ the extremely stable hydrogen bond between Asp90 and Lys92 ob- A. (1992) Mg confers DNA binding specificity to the EcoRV restriction endonuclease. Biochemistry 31, 3727–3732. served in 1TA would render the acceptance of a proton by Asp90, [7] Taylor, J.D., Badcoe, I.G., Clarke, A.R. and Halford, S.E. (1991) EcoRV restriction and thus the activation of the nucleophile, unlikely. Such hindered endonuclease binds all DNA sequences with equal affinity. Biochemistry 30, nucleophile formation might inhibit catalysis. The present results 8743–8753. [8] Engler, L.E., Welch, K.K. and Jen-Jacobson, L. (1997) Specific binding by EcoRV are consistent with the notion that changes in the charge distribu- endonuclease to its DNA recognition site GATATC. J. Mol. Biol. 269, 82–101. tion at the catalytic site induce a rearrangement of the catalytic [9] Sidorova, N.Y., Muradymov, S. and Rau, D.C. (2011) Solution parameters site groups [27]. modulating DNA binding specificity of the restriction endonuclease EcoRV. FEBS J. 278, 2713–2727. [10] Pingoud, A., Fuxreiter, M., Pingoud, V. and Wende, W. (2005) Type II restriction 4. Conclusions endonucleases: structure and mechanism. Cell. Mol. Life Sci. 62, 685–707. [11] Pingoud, V., Wende, W., Friedhoff, P., Reuter, M., Alves, J., Jeltsch, A., Mones, L., Fuxreiter, M. and Pingoud, A. (2009) On the divalent metal ion dependence of The mechanism of DNA hydrolysis reactions in metallonucleas- DNA cleavage by restriction endonucleases of the EcoRI family. J. Mol. Biol. es has been the subject of much debate [28–31]. The present sim- 393, 140–160. ulations of EcoRV–DNA complexes show a clear effect on the [12] Horton, N.C., Newberry, K.J. and Perona, J.J. (1998) Metal ion-mediated 2+ substrate-assisted catalysis in type II restriction endonucleases. Proc. Natl. conformation of the complex of Mg deletion at the B site. The Acad. Sci. USA 95, 13489–13494. DNA loses the 50° kink at the central TA step characteristic of a [13] Baldwin, G.S., Sessions, R.B., Erskine, S.G. and Halford, S.E. (1999) DNA cleavage-ready complex. The loss of strong hydrogen bonds, the cleavage by the EcoRV restriction endonuclease: roles of divalent metal ions in specificity and catalysis. J. Mol. Biol. 288, 87–103. overall reduction of the hydrogen-bond occupancies and the less [14] Horton, N.C. and Perona, J.J. (2004) DNA cleavage by EcoRV endonuclease: two favorable protein–DNA interaction energy indicate that the pro- metal ions in three metal ion binding sites. Biochemistry 43, 6841–6857. tein–DNA interaction is significantly weakened in 1TA. The change [15] Groll, D.H., Jeltsch, A., Selent, U. and Pingoud, A. (1997) Does the restriction endonuclease EcoRV employ a two-metal-ion mechanism for DNA cleavage? of the hydrogen-bond pattern in 1TA can be traced back to a struc- Biochemistry 36, 11389–11401. tural change in the active site. [16] Imhof, P., Fischer, S. and Smith, J.C. (2009) Catalytic mechanism of DNA The present findings suggest that Metal B serves to stabilize a backbone cleavage by the restriction enzyme EcoRV: a quantum mechanical/ distorted DNA conformation, i.e. a sharp kink at the cleavage re- molecular mechanical analysis. Biochemistry 48, 9061–9075. [17] Mones, L., Simon, I. and Fuxreiter, M. (2007) Metal-binding sites at the active gion, thereby allowing the formation of a tight complex in which site of restriction endonuclease BamHI can conform to a one-ion mechanism. the reaction partners are poised for the hydrolysis to occur. Biol. Chem. 388, 73–78. [18] Mackerell, A.D. and Nilsson, L. (2008) Molecular dynamics simulations of nucleic acid–protein complexes. Curr. Opin. Struct. Biol. 18, 194–199. Acknowledgments [19] Zahran, M., Daidone, I., Smith, J.C. and Imhof, P. (2010) Mechanism of DNA recognition by the restriction enzyme EcoRV. J. Mol. Biol. 401, 415–432. M.Z. thanks Loukas Petridis, Karine Voltz and Isabella Daidone [20] Guroult, M., Picot, D., Abi-Ghanem, J., Hartmann, B. and Baaden, M. (2010) How cations can assist DNase I in DNA binding and hydrolysis. PLoS Comput. for their useful comments on the paper and Thomas Splettstoesser Biol. 6, e1001000. for his help on the figures. P.I. is grateful for funding from the Hei- [21] Hiller, D.A., Rodriguez, A.M. and Perona, J.J. (2005) Non-cognate enzyme–DNA delberg BIOMS initiative. J.C.S. was funded by a Laboratory-Direc- complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC. J. Mol. Biol. 354, 121–136. ted Research and Development Grant from the US Department of [22] Dickerson, R.E. (1998) DNA bending: the prevalence of kinkiness and the Energy. This research was supported in part by the National Sci- virtues of normality. Nucleic Acids Res. 26, 1906–1926. ence Foundation through TeraGrid resources provided by NICS. [23] El Hassan, M.A. and Calladine, C.R. (1996) Propeller-twisting of base-pairs and the conformational mobility of dinucleotide steps in DNA. J. Mol. Biol. 259, 95– 103. Appendix A. Supplementary data [24] Horton, N.C. and Perona, J.J. (2000) Crystallographic snapshots along a protein- induced DNA-bending pathway. Proc. Natl. Acad. Sci. USA 97, 5729–5734. [25] Stoever, T., Khler, E., Fagin, U., Wende, W., Wolfes, H. and Pingoud, A. (1993) Supplementary data associated with this article can be found, in Determination of the DNA bend angle induced by the restriction endonuclease the online version, at doi:10.1016/j.febslet.2011.07.036. EcoRV in the presence of Mg2+. J. Biol. Chem. 268, 8645–8650. [26] Selvaraj, S., Kono, H. and Sarai, A. (2002) Specificity of protein–DNA recognition revealed by structure-based potentials: symmetric/asymmetric References and cognate/non-cognate binding. J. Mol. Biol. 322, 907–915. [27] Horton, N.C., Otey, C., Lusetti, S., Sam, M.D., Kohn, J., Martin, A.M., [1] Kovall, R.A. and Matthews, B.W. (1999) Type II restriction endonucleases: Ananthnarayan, V. and Perona, J.J. (2002) Electrostatic contributions to site structural, functional and evolutionary relationships. Curr. Opin. Chem. Biol. 3, specific DNA cleavage by EcoRV endonuclease. Biochemistry 41, 10754– 578–583. 10763. [2] Winkler, F.K., Banner, D.W., Oefner, C., Tsernoglou, D., Brown, R.S., Heathman, [28] Syson, K., Tomlinson, C., Chapados, B.R., Sayers, J.R., Tainer, J.A., Williams, N.H. S.P., Bryan, R.K., Martin, P.D., Petratos, K. and Wilson, K.S. (1993) The crystal and Grasby, J.A. (2008) Three metal ions participate in the reaction catalyzed structure of EcoRV endonuclease and of its complexes with cognate and non- by T5 flap endonuclease. J. Biol. Chem. 283, 28741–28746. cognate DNA fragments. EMBO J. 12, 1781–1795. [29] Mueser, T.C., Nossal, N.G. and Hyde, C.C. (1996) Structure of bacteriophage T4 [3] Chandrasekaran, R. and Arnott, S. (1996) The structure of B-DNA in oriented RNase H, a 50 to 30 RNA–DNA and DNA–DNA exonuclease with sequence fibers. J. Biomol. Struct. Dyn. 13, 1015–1027. similarity to the RAD2 family of eukaryotic proteins. Cell 85, 1101–1112. [4] Sissi, C. and Palumbo, M. (2009) Effects of magnesium and related divalent [30] Dupureur, C.M. (2008) Roles of metal ions in nucleases. Curr. Opin. Chem. Biol. metal ions in topoisomerase structure and function. Nucleic Acids Res. 37, 12, 250–255. 702–711. [31] Dupureur, C.M. (2010) One is enough: insights into the two-metal ion [5] Mones, L., Kulhnek, P., Florin, J., Simon, I. and Fuxreiter, M. (2007) Probing the nuclease mechanism from global analysis and computational studies. two-metal ion mechanism in the restriction endonuclease BamHI. Metallomics 2, 609–620. Biochemistry 46, 14514–14523.