Unveiling A Single-Metal Mediated Phosphodiester Bond Cleavage

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Unveiling A Single-Metal Mediated Phosphodiester Bond Cleavage Mechanism for Nucleic Acids: A Multiscale Computational Investigation of a Human DNA Repair Enzyme Mohamed M. Aboelnga, and Stacey D Wetmore J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b03986 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 3, 2019

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Journal of the American Chemical Society

Unveiling A Single-Metal Mediated Phosphodiester Bond Cleavage Mechanism for Nucleic Acids: A Multiscale Computational Investigation of a Human DNA Repair Enzyme Mohamed M. Aboelnga and Stacey D. Wetmore* Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge, Alberta, Canada T1K 3M4

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ABSTRACT: Despite remarkable stability, the phosphodiester bond of nucleic acids is hydrolytically cleaved in critical biological processes. Although this reaction is commonly accepted to take place via a twometal assisted mechanism, recent experimental evidence suggests that several enzymes use a single metal ion, but the precise catalytic mechanism is unknown. In the present work, we employ a multiscale computational approach to decipher the phosphodiester cleavage mechanism for this unique pathway by focusing on the human APE1 repair enzyme, which catalyzes the incision of phosphodiester bonds adjacent to DNA lesions. To resolve ambiguity in the literature regarding the role of the single metal (Mg(II)) center, several catalytic mechanisms were carefully examined. Our predicted preferred hydrolysis pathway proceeds in two steps via a pentacovalent phosphorane intermediate in the absence of substrate ligation to Mg(II), with a rate-limiting barrier (19.3 kcal/mol) in close agreement with experiment (18.3 kcal/mol). In this mechanism, D210 promotes catalysis by activating water for nucleophilic attack at the 5′-phosphate group with respect to the damaged site. Subsequently, a Mg(II)-bound water triggers leaving group departure by neutralizing the 3′-hydroxyl of the neighboring nucleotide. Consistent with experimental kinetic and mutational data, several other active site residues (N212, Y171 and H309) play multiple roles throughout the reaction to facilitate this challenging chemistry. In addition to revealing previously unknown mechanistic features of the APE1 catalyzed reaction, our work sets the stage for exploring the phosphodiester bond cleavage catalyzed by other single-metal dependent enzymes, as well as different pharmaceutical and biotechnological applications.


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INTRODUCTION Phosphodiester bonds are the backbone linkages of all nucleic acids. The uncatalyzed scission of a nucleic acid backbone is kinetically challenging, with an estimated half-life of 30 million years under mild conditions.1 Despite this remarkable resistance to hydrolysis, the cleavage of phosphodiester bonds is central to many critical biological processes, including DNA replication and repair, as well as RNA processing and degradation.2 These processes are accelerated to life sustainable rates by nucleases,3 which facilitate hydrolysis in many ways including stabilizing and positioning the negatively charged substrate, and promoting the departure of the leaving group.4 Although the identity of the base that activates the water nucleophile is a controversial issue for many nucleases,5-8 it is well established that the most common catalytic pathway is a two-metal ion dependent mechanism in which each metal plays a distinct role.2, 4, 9-11 Specifically, the first metal acts as a Lewis acid to lower the pKa of the nucleophilic water and thereby helps the base initiate the reaction, while the second metal stabilizes the charge of the leaving group through direct ligation.9 Nucleases that target DNA are particularly interesting since the phosphodiester bond in DNA is 400 times more stable than in RNA.12 In addition to the universal two-metal assisted pathway, recent studies have clarified that some DNA nucleases utilize three-metal ions.13-15 Moreover, a single-metal ion dependent mechanism has been proposed for many DNA nucleases.16-17 Interestingly, evidence to date suggests the metal ion used by single-metal dependent nucleases is not properly positioned to activate the nucleophilic water, but instead likely aids leaving group stabilization through its hydration sphere or direct ligation.18-22 Indeed, a metal is equivalently positioned in close proximity to the leaving group in one-metal and two-metal dependent nucleases.17 Nevertheless, Mg(II) is the native metal in all two-metal dependent



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nucleases, while one-metal dependent DNA nucleases are less stringent on the identity of the metal ion.9, 16-17 Although the chemistry of two-metal assisted phosphodiester hydrolysis has been the subject of many computational studies for a variety of nucleases,7, 23-25 the atomistic details of the novel chemistry facilitated by single-metal dependent nucleases are poorly understood. Therefore, the principal aim of this study is to provide a solid understanding of the phosphodiester cleavage mechanism for a nuclease that has a single metal cofactor. Human apurinic/apyrimidinic (AP) endonuclease (APE1) is one of the most experimentally investigated single-metal dependent endonucleases.26 APE1 is a ubiquitous DNA repair enzyme that participates in base excision repair (BER), with the main role to remove cytotoxic abasic sites.27 Abasic sites are known to be the most abundant DNA lesions and are major threats to genome stability.28 Therefore, the function of APE1 is essential for genetic integrity.29 APE1 selectively recognizes abasic sites from canonical nucleotides and flips the lesions out of the DNA helix into the active site to initiate repair.30 The enzyme also catalyzes the hydrolysis of the 5′phosphodiester backbone with respect to the abasic site, generating a single-strand break with 3′hydroxy and 5′-deoxyribose phosphate (5′-dRP) termini. A wealth of experimental data on APE1 has provided valuable insight regarding its structure and function. Although the number and identity of the divalent metal cofactors in the APE1 active site was initially a long-standing debate,30-34 recent structural studies confirm that APE1 is a single Mg(II)-dependent enzyme.35-36 Furthermore, key active site residues have been identified (Figure 1) and different catalytic functions proposed.33, 35, 37-38 Specifically, based on mutational data and close proximity to the phosphate backbone, Y171 was originally proposed to initiate the reaction through nucleophilic attack,39 a suggestion later rejected for a proposal that Y171 most likely acts as a guide to properly position the substrate for catalysis.40 Indeed, a Y171F 4 ACS Paragon Plus Environment

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mutation results in a 1,200-fold decrease in the catalytic rate of APE1.38, 41 Although D210 has been proposed to neutralize the leaving group,37 a D210N mutation has the most profound impact on the reaction rate, inducing a 10,000 fold reduction.41 Therefore, mutational data is likely more consistent with the proposal that D210 acts as the base that activates the nucleophilic water.40-42 In addition to D210, both N212 and H309 significantly impact the catalytic rate, with N212A and H309N mutations leading to 7,000 and 2,500 fold decreases, respectively. N212 has been suggested to position D210 or the nucleophilic water,43 while H309 has been proposed to activate the nucleophilic water44-45 or stabilize the negative charge accumulating on the backbone as the reaction proceeds.32, 37, 46-48 Despite the valuable information obtained to date about essential active site residues, the identity of the ligands coordinated to the Mg(II) ion in the catalytically active form of APE1 is a controversial issue.36, 38, 44, 46 This uncertainty arises due to the fact that X-ray structures are only available for the apoenzyme,35 the APE1–DNA product complex30, 38, 41, 48 and a Mn(II)-containing complex bound to a phosphorothioate DNA analogue.38 In the Mg(II)-containing product complex (Figure 1A), the metal ion has an octahedral coordinate sphere, the preferred coordination sphere for Mg(II).49 Specifically, Mg(II) is directly coordinated to three water molecules, the carboxylate side chain of E96, and the 3′-OH and 5′-dRP termini of the incised abasic substrate.30,

38, 41

However, short (15 ns) molecular dynamics (MD) simulations initiated from a model developed by reverting the product structure to a reactive complex suggest no direct ligation between the phosphate oxygen and Mg(II), with the octahedral geometry around the metal fulfilled by D70.41 This proposal is supported by an X-ray structure of an APE1–DNA reactive complex analogue that contains Mn(II) and phosphorothioate (Figure 1B).38 Specifically, this snapshot demonstrates that Mn(II), which is known to behave similar to Mg(II),50 coordinates to the carboxylate side 5 ACS Paragon Plus Environment


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chains of both D70 and E96, as well as four water molecules, but not the substrate. The ligation of both D70 and E96 to the divalent metal is further supported by a high-resolution X-ray structure of the Mg(II)-containing apoenzyme.35 Nevertheless, the absence of direct ligation between the thiosubstituted abasic substrate and the metal may be at least partially due to the substitution of DNA backbone oxygen atoms with sulfur atoms,51 while the impact of substrate binding on the crystallized apoenzyme structure is unclear. Importantly, the absence of ligation between D70 and Mg(II) in all crystallized product complexes has been suggested to be due to movement of Mg(II) after the chemical step, which does not affect coordination to E96 due to the flexibility of this residue.32, 35, 52 The current study strives to clarify uncertainties surrounding the phosphodiester bond cleavage mechanism catalyzed by APE1, and thereby resolve current debates in the literature regarding the function of nucleases that depend on a single metal ion. Specifically, different binding configurations surrounding the metal ion and different roles for key APE1 active site residues are initially scrutinized using quantum mechanical (QM) models, which have been proven to be powerful tools for exploring the catalytic mechanisms of biosystems, including metalloenzymes.53-54 Subsequently, the details of the most probable pathway are further explored with MD and quantum mechanics/molecular mechanics (QM/MM) approaches. Our data clarifies the catalytic activity of this critical BER enzyme toward abasic sites. This new knowledge can be applied to better understand other catalytic functions of APE1. Indeed, APE1 is also known to use the same active site to catalyze many other processes including DNA exonuclease activity toward mispaired DNA,55-56 nucleotide incision repair (NIR) of oxidative nucleobase damage,57-58 and endoribonuclease activity.59-60 Moreover, APE1 has been proposed to be a valid drug target for enhancing the effectiveness of existing chemotherapies since overexpression of APE1 has been 6 ACS Paragon Plus Environment

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reported in many drug-resistant tumors.61-62 Indeed, many small molecules have been identified as candidates for inhibiting APE1 activity in tumor cells.63 Furthermore, the mechanistic details for a one-metal dependent nuclease characterized for the first time in the present work may be more broadly applicable to other DNA nucleases and other classes of enzymes that share similar active sites (for example, the sequence similarity between the active site of APE1 and human tyrosylDNA phosphodiesterase suggests a shared catalytic pathway).64 Finally, this work will aid metalloprotein engineering for which there is currently an abundance of interest.65-66

COMPUTATIONAL METHODS Quantum Mechanical Calculations: QM-cluster models were initially used to explore the mechanistic details associated with different active site configurations. Two main chemical architectures of the APE1 active site were considered, which were built from X-ray structures to ensure proper placement of key active site residues with respect to the substrate. Specifically, the substrate is directly ligated to the Mg(II) ion in chemical architecture I, which was built from APE1 product complexes (PDB ID: 4IEM; 2.39 Å resolution; Figure 1A).41 In contrast, an additional water molecule rather than the substrate is ligated to the metal ion in chemical architecture II, which was built based on the Mn(II)-containing APE1–DNA thiosubstrate complex (PDB ID: 5DG0; 1.8 Å resolution; Figure 1B).38 For both architectures, three orientations of D70 with respect to the Mg(II) coordination sphere were considered due to differences in D70 coordination in the crystal structures (Figure 1). All QM-cluster models include the active site residues previously proposed to directly participate in the catalytic mechanism by activating the water nucleophile, stabilizing the transition state or aiding leaving group departure (namely D70, E96, Y171, D210, N212 and H309) or may position these residues (namely N68 and N174). Each amino 7 ACS Paragon Plus Environment


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acid was truncated at the -carbon, with the exception of E96, which was truncated at the -carbon of the side chain. A DNA substrate was used that includes the abasic site truncated at O3′, and the sugar from the 5′-adjacent nucleoside truncated at O5′ and the associated base replaced with an amino group. Together, these choices resulted in models with a total charge of –1, and 153 and 156 atoms for architectures I and II, respectively, which satisfies the model size proven to be required for a reasonable representation of an active site.53 Due to previous successes in modeling similar enzymatic systems,67-68 the M06-2X69 functional in conjunction with 6-31G(d,p) as implemented in Gaussian 09 (revision D.01) was used to characterize the pathways.70 Relative Gibbs energies that include thermal corrections were determined using IEFPCM71-M06-2X/6311+G(2df,p) with a dielectric constant of ε=4 (see Table S1 in the Supporting Information for a comparison of relative energies with and without the thermal corrections). Molecular Dynamics Simulations: The binding geometry that led to the most enzymatically feasible hydrolysis mechanism according to the QM-cluster calculations was modeled in the APE1–DNA complex using MD simulations to further explore the active site structure. The MD model was built from the crystal structure for the APE1–DNA product complex containing the 3′OH and 5′-dRP groups directly ligated to the Mg(II) ion (PDB ID: 4IEM).41 Additional simulations were performed from models built from an alternative crystal structure for the APE1–DNA product complex (PDB ID: 5DFF) and reactant analogue (PDB ID: 5DG0).38 AMBER ff14SB parameters were assigned to the entire APE1–DNA complex, while AMBER ff14SB parameters were supplemented by GAFF parameters for the nonstandard abasic site using the ANTECHAMBER module of AMBER 14.72 The enzyme–DNA system was neutralized by adding Na+ ions and solvated in a TIP3P octahedral explicit water box. The entire system was then minimized, heated


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and submitted for a series of equilibration steps. Using the periodic boundary condition, an unconstrained 2.5 s production simulation was performed. Quantum Mechanics/Molecular Mechanics Calculations: To further explore the catalytic mechanism characterized using QM-cluster calculations, the reaction was further explored using the ONIOM formalism with mechanical embedding,73-74 which has proven to be a powerful tool for characterizing the catalytic mechanism of many repair enzymes,75-77 as well as other enzymes.78 Specifically, a model of the entire solvated APE1–DNA complex (9445 atoms) was built based on a representative structure from the MD simulations chosen according to the root-mean-square deviations (RMSD) of key active site residues. Water more than 4 Å from the edge of the APE1– DNA system was removed from the representative structure and the new system MM (AMBER ff14SB) minimized. The D70, E96, Y171, N174, D210, N212, and H309 residues, the AP site and five water molecules (including the nucleophilic water) form the QM region (121 atoms), which was treated with M06-2X/6-31G(d,p).70 The remainder of the system was included in the MM layer and treated by AMBER (ff14SB). For all stationary points, all atoms in the model were optimized (including the whole enzyme, DNA and water). Frequency calculations were performed to characterize stationary points and determine the thermal energy corrections used to calculate the Gibbs

energies.

Relative

energies

were

evaluated

using

ONIOM(M06-2X/6-

311+G(2df,p):AMBER14SB). ONIOM calculations were performed using Gaussian 09 (revision D.01).70 Full details of the computational methodology are provided in the Supporting Information (SI).



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RESULTS AND DISCUSSION Effect of the Mg(II) Coordination Geometry. Due to the current debate regarding the ligands involved in the octahedral coordination sphere around the single Mg(II) ion in the APE1 active site,36,

38, 44, 46

various QM-cluster models were used to examine the effect of the metal ion

coordination geometry on the hydrolysis pathway. This is critical because it has been shown that the geometry of the coordination sphere around Mg(II) significantly contributes to the overall hydrolysis activity of similar Mg(II)-dependent phosphatases.79 As described in the Computational Methods, experimental structures (Figure 1) were used to build two chemical architectures (Figures 2 and 3), which differ in whether the substrate is (architecture I) or is not (architecture II) ligated to Mg(II). For each architecture, three configurations were considered that have unique orientations of D70 with respect to Mg(II) (Figures 2 and 3). Specifically, in configuration a, D70 is not ligated to the metal ion in correlation with crystal structures of the APEI–DNA product complex (Figure 1A).41 In configurations b and c, D70 is coordinated to Mg(II) as per the crystal structure of APE1 bound to a phosphothionate substrate in the presence of Mn(II) (Figure 1B),38 as well the crystallized apoenzyme.35 However, these latter two configurations differ in the absence (b) or presence (c) of hydrogen bonding between the unligated carbonyl of D70 and a neighboring coordinated water molecule (Figures 2 and 3). In all cases, the preferred octahedral coordination sphere of Mg(II) is fulfilled by E96 and water molecules. Regardless of the active site conformation (architecture I or II coupled with configuration a, b or c), our QM-cluster calculations predict a two-step hydrolysis mechanism for the phosphodiester bond cleavage facilitated by APE1 (Scheme 1). In particular, D210 acts as the base that deprotonates a nucleophilic water positioned in close proximity to the DNA backbone, which promotes simultaneous nucleophilic attack on the substrate phosphorus center in the first transition 10 ACS Paragon Plus Environment

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structure. This yields a phosphorane intermediate that has similar structural features to those characterized for the two-metal mediated phosphodiester bond cleavage facilitated by other enzymes.23, 80 In the second reaction step, a nearby metal-activated water molecule is poised to aid leaving group departure and transfers a proton to the substrate to yield a product that contains a cleaved phosphodiester bond. The distinct structural features and the relative importance of the six active site conformations will be discussed in the following sections. (i) Substrate Directly Ligated to the Metal Ion (Architecture I). In architecture I, the nonbridging phosphate oxygen of the substrate is directly ligated to Mg(II) (Figure 2A). Regardless of the orientation of D70 relative to the Mg(II) coordination sphere, the calculated rate-limiting barrier for architecture I (up to 38.7 kcal/mol, Figure 2B) is significantly larger than the experimentally predicted barrier (18.3 kcal/mol),38 and the reaction is endothermic (by up to 23.5 kcal/mol, Figure 2B). The major reasons for the unfavorable reaction energetics are the orientations of H309 and Y171. In fact, ligation of the substrate to Mg(II) in the reactive complex disrupts the crystal structure orientation of H309, as well as Y171, which have both been shown to significantly contribute to catalysis.37, 41-42 Specifically, in all optimized reactive complexes (IRC), cationic H309 is directed away from the DNA phosphate backbone and interacts with the nucleophilic water molecule (1.53–1.66 Å, Figure S1). This contrasts H309 being directed toward the cleaved phosphodiester backbone in the crystal structure of a product complex41 (Figure 1A) since the cleaved backbone requires more stabilization from H309 than the intact bond in IRC. Although H309 is also directed toward the phosphate backbone in the crystallized reactant analogue that lacks a direct substrate–metal interaction (Figure 1B),38 charge stabilization afforded through ligation of the phosphate backbone to Mg(II) in our model (IRC) eliminates the need for stabilization by H309 and leads to active site reorganization. Similarly, direct ligation of the abasic 11 ACS Paragon Plus Environment


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substrate to Mg(II) in architecture I prevents an interaction between the Y171 hydroxyl group and the substrate that is inferred from crystal structures (Figure 1),38, 41, 48 with the Y171 side chain instead forming a hydrogen bond (H-bond) with E96 in IRC (1.85–2.04 Å, Figure S1). Upon nucleophilic attack on the phosphodiester backbone in the first transition state (ITS1, Figure S1), H309 repositions toward the substrate backbone and maintains backbone interactions throughout the remainder of the reaction pathway, which is more consistent with the placement of this residue with respect to the substrate in crystal structures.38,

41

The negative charge

accumulating on the substrate backbone along the reaction is stabilized by ligation to the metal and protonation of a nonbridging backbone oxygen atom by H309. Nevertheless, these features do not sufficiently offset the decreased nucleophilicity of water due to hydrogen bonding with cationic His309 in the reactive complex and the energetic cost to rearrange H309. Furthermore, Y171 maintains H-bonding interactions with E96 throughout the reaction (Figure S1) rather than being close to the substrate as observed in experimental X-ray structures (Figure 1),30, 38, 41 and therefore does not directly participate in the reaction in a way that explains the reported catalytic impact.4041

In the case of configuration a, the Mg(II) coordination sphere is also disrupted, with E96

adopting a monodentate binding configuration. Overall, the inconsistency of architecture I with experimental kinetic,40 mutational41 and structural data30, 38, 41 suggests that the substrate is unlikely coordinated to Mg(II) in the reactive complex and throughout the chemical step. (ii) Substrate Unligated to the Metal Ion (Architecture II). The octahedral binding geometry around Mg(II) in architecture II is satisfied by four water molecules and the side chains of E96 and D70 (Figure 3A). Similar to architecture I (substrate ligated to the metal), three configurations around the Mg(II) ion were modeled that vary in the orientation of D70 (Figure 3A). Unlike for architecture I, H309 and Y171 are within H-bonding distance of the substrate backbone throughout 12 ACS Paragon Plus Environment

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the pathway regardless of the configuration for architecture II (Figure S2), which is consistent with the locations of these residues in crystal structures.30, 38, 41, 48 For configuration a, the Gibbs energy barriers (26.5 and 24.1 kcal/mol, Figure 3B) are significantly smaller than those obtained for the same configuration of architecture I (35.8 and 38.7 kcal/mol, Figure 2B). This barrier reduction occurs due to the persistent H-bonding interactions between cationic H309 and Y171 and the phosphate backbone, as well as maintenance of the octahedral binding geometry around Mg(II), throughout the reaction pathway. Nevertheless, the bidentate ligation of E96 to Mg(II) in configuration a results in a constrained geometry of the metal center. Moreover, there is an energetic cost associated with reorientation of N174 to interact with the nonbridging oxygen of the substrate in IITS1a, a position maintained throughout the remainder of the reaction (Figure S3). Together, these factors lead to a higher overall barrier for this active site configuration (26.5 kcal/mol) than experimentally predicted (18.3 kcal/mol).38 Upon optimization, the Mg(II) coordination geometry of configuration b collapses to configuration c, which is consistent with literature suggesting that the unligated oxygen of an Asp/Glu coordinated to Mg(II) preferentially H-bonds with a neighboring ligated water molecule.81 For configuration c, a two-step hydrolysis mechanism was successfully characterized (Figures 3 and S2), which maintains a preferred Mg(II) octahedral binding geometry that is less constrained than configuration a due to the lack of bidentate ligation. When coupled with stabilization of the first transition state through protonation by H309 and tighter H-bonding with Y171 (i.e., decreases by 0.27–0.28 Å), the barrier for the first step is 16.7 kcal/mol. Furthermore, the second (rate-limiting) barrier is much less for this active site geometry (22.5 kcal/mol) than any other configuration and the product lies only 2.8 kcal/mol above the corresponding reactive complex (Figure 3B). 13 ACS Paragon Plus Environment


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Overall, chemical architecture II coupled with configuration c results in the lowest calculated barriers and most thermodynamically favorable product amongst all chemical architectures and configurations considered (Figures 2 and 3). Direct ligation of the substrate to Mg(II) (architecture I) is unfavorable at least in part due to the poor alignments of H309 and Y171 in the reactive complex, while configuration a is less desirable due to the highly constrained Mg(II) coordination sphere. We note that our calculated product complex geometry differs from those experientially crystallized (Figure 1A),30,

38, 41, 48

mainly due to the reported disordering and

plasticity of the Mg(II) binding domain.32, 35, 52 Indeed, the repositioning of Mg(II) to bind to the 3′-OH and 5′-dRP leaving groups of the cleaved abasic site observed in the crystallized product complexes is indicative of a slow step that occurs after the chemical step modelled in the present study.42,56 The most favorable active site arrangement (architecture II combined with configuration c, Figure 3) will be used to further probe features of the APE1 catalyzed reaction in the subsequent sections.

Roles of H309 and Y171. Since a H309N mutation results in a 2,500-fold decrease in the catalytic rate,41 H309 in the neutral form has been proposed to act as the base that initiates the phosphodiester bond cleavage.40, 44 When this pathway is considered using the preferred QMcluster model, both neutral H309 and D210 hydrogen bond to the nucleophilic water in the reactive complex (Figure S4A). The substrate backbone is partially charge stabilized through hydrogen bonds with Y171, as well as two Mg(II)-coordinated water molecules. However, these interactions are insufficient to offset the increased charge in a phosphorane intermediate. Therefore, the reaction with neutral H309 acting as the base occurs in a single step, with a high energy barrier (36.6 kcal/mol, Figure S5). When coupled with the calculated orientation of neutral H309 (Figure 14 ACS Paragon Plus Environment

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S4A) being inconsistent with crystal structures30, 38, 41 (Figure 1) and the more favorable barriers obtained in the present work when D210 activates the water nucleophile, H309 is unlikely the base for the APE1-catalyzed bond cleavage. Furthermore, when D210 activates the water nucleophile and neutral H309 is in the active site, a similar single-step, high energy (38.3 kcal/mol) pathway is characterized (Figures S4B and S5). Thus, although H309 has been previously suggested to be neutral and initiate the reaction by activating a water nucleophile,40, 44 our calculations clearly demonstrate that H309 must be protonated in order to help neutralize the charge on the phosphorane intermediate, which is in good agreement with NMR and crystallographic data.38, 41, 45

Although mutation of D210 or H309 more significantly impacts the catalytic power of APE1, the Y171F mutation leads to a 1,200-fold decrease in the catalytic rate.41 However, there is a debate in the literature regarding whether this effect arises from Y171 acting as the nucleophile.3940

To better understand the role of Y171, the reaction pathway was mapped for the Y171F mutant

(Figure S6). The general two-step mechanism is maintained and the overall structural features are similar for the mutant and wild-type models (Figures S2, S6 and S7). Additionally, the barrier for the rate-determining step for the mutant enzyme (Figure S5) is 3.4 kcal/mol larger than that for the wild-type enzyme (Figure 3). The small difference in the predicted barriers is fully consistent with the reported 1200-fold decrease in the catalytic rate upon Y171 mutation.41 When coupled with the similar two-step mechanisms for the Y171F mutant and wild-type models, our calculations suggest that Y171 does not play a mechanistic role in the overall chemical reaction, such as acting as the base. This conclusion aligns with more recent proposals in the literature that Y171 is oriented to interact with the substrate.40 In fact, our calculations reveal that a H-bond between Y171 and the substrate provides subtle stabilization of the negative charge developing on the backbone as 15 ACS Paragon Plus Environment


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the reaction proceeds. This proposed role is consistent with the smaller mutational effect on the catalytic rate for Y171 compared to H309.41 Regardless of the roles proposed in the present section for H309 and Y171, the contributions of both residues to the mechanism will be further explored by considering a more complete APE1–DNA model in the following section.

Impact of the Surrounding Enzyme Environment on the Phosphodiester Bond Cleavage Mechanism. To further investigate the phosphodiester bond cleavage mechanism facilitated by APE1, the preferred Mg(II) binding configuration determined from the QM-cluster calculations was investigated in an APE1–DNA complex. Initially, classical MD simulations were employed to understand the impact of the predicted Mg(II)-coordination sphere on the active site configuration. Regardless of the crystal structure used to build the APE1–DNA reactant complex or the simulation length, the preferred octahedral coordination geometry was maintained for Mg(II), which involves D70, E96 and four water molecules (Figures S8–S9). A strong H-bond between cationic H309 and the substrate backbone (average distance: 1.79±0.12 Å) also persists, which results in a H309 orientation consistent with crystal structures38, 41 (Figure S10) and further underscores the conclusion that H309 is cationic. Moreover, the position of Y171 matches that in experimental X-ray structures (Figure S10B–C). In this orientation, Y171 participates in a Hbonding network with the nucleophilic water, which results in a water-mediated H-bonding interaction with the backbone (Figure S10A). Importantly, rather than directly interacting with the nucleophile as previously suggested,41 N212 maintains a H-bond with D210 (r(D210COO−···H– NN212) = 1.96±0.19 Å), which properly positions D210 in the active site. Indeed, a water molecule H-bonds with D210 (r(D210COO−···H–Owater) = 1.71±0.14 Å) and adopts an appropriate position


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for nucleophilic attack (average distance: 3.42±0.12 Å). Thus, the APE1–DNA complex maintains a catalytically active configuration throughout the MD simulations. Subsequently, a QM/MM model (Figure 4) was built from an MD representative structure. In the QM/MM optimized reactive complex (Figure 5), the Mg(II) ion is coordinated to D70, E96 and four water molecules (average distance = 2.06±0.02 Å) as per experimental X-ray structures of the apoenzyme35 and substrate analogue.38 A nucleophilic water molecule is correctly positioned in close proximity to the DNA backbone (r(P···Owater) = 3.07 Å) for nucleophilic attack through tight hydrogen bonds with D210 (r(D210COO···H–Owater) = 1.86 Å) and Y171 (r(Y171O···Hwater) = 1.95 Å, Figures 5 and S9). D210 is held in this critical position through a moderately tight H-bond with the amide side chain of nearby N212 (r(D210COO···H–NN212) = 2.05 Å), which adopts a position in agreement with crystal structures.38, 41 The abasic substrate is properly oriented for nucleophilic attack through H-bonding with H309 (r(O···HH309) = 1.63 Å) and a Mg(II)coordinated water (r(O3′···H–Owater) = 1.98 Å), interactions that will also serve to stabilize the charge developing on the backbone as the reaction proceeds. In the first transition state (TS1), a proton from water is shifted to D210 (1.25 Å), and the H-bond between D210 and N212 lengthens as the nucleophilic water moves closer to the phosphate group of the abasic substrate (r(P···Owater) = 2.01 Å). Throughout this process, H309 and Y171 are in close proximity to the backbone as per X-ray crystal structures.38, 41 However, the hydroxy group of Y171 aids charge stabilization in the transition state by forming a H-bond with the nucleophile (r(Y171OH···Owater) = 1.94 Å) rather than the substrate. Relative to the QM-cluster reactive complex (IIRCc, Figure S2), the abasic site is positioned in a more productive orientation for bond cleavage (Figure S11) and thus less energy is required to reach the first transition state (15.6 kcal/mol, Figure 5). 17 ACS Paragon Plus Environment


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The first transition state is connected to a stable phosphorane intermediate complex (IC, Figure 5 and S9) that has similar features to those characterized for the two-metal mediated phosphodiester bond cleavage facilitated by other enzymes.23,

80

In IC, the proton is fully

transferred from water to D210, a new Owater__P single bond is formed (1.64 Å), and the P···O3′ bond is slightly elongated (1.78 Å, Figure 5 and S9). Similar to TS1, the phosphorane intermediate is stabilized by cationic H309 and hydrogen bonding to a Mg(II)-ligated water molecule. However, both Y171 and N212 are now also positioned to H-bond with the backbone (r(Y171H···O) = 1.86 Å and r(N212H···O) = 1.93, Figure S11) and thereby provide additional stabilization to the intermediate. As a result, the IC falls 16.6 kcal/mol above the reactive complex. The only acidic residue poised to aid bond cleavage in IC is a nearby metal-activated water molecule (r(O3′···Hw1) = 1.61 Å, Figure 5 and S9). The position of this Mg(II)-bound water together with its lower pKa facilitates proton transfer to O3′ of the leaving group through the second transition state (TS2), with an energy barrier of 19.3 kcal/mol (Figure 5). In ITS2, the shifted proton is 1.10 Å from O3′, while the P···O3′ bond is partially cleaved (2.15 Å). The collapse of TS2 leads to the product complex (PC) in which the phosphodiester bond is completely cleaved (r(P···O3′) = 2.86 Å) and the 3′-O leaving group is fully neutralized. The PC for the APE1–DNA model is thermodynamically stable, lying 17.7 kcal/mol below the corresponding RC (Figure 5). Furthermore, comparison of the calculated and crystallized38, 41 product complexes reveals alignment of key active site residues (Figure S12). In fact, deviations mainly occur in the position of Mg(II) and the orientation of the E96 side chain, which have both been experimentally reported to be highly flexible after the chemical step.32, 35, 38, 56 Overall, our predicted rate-limiting barrier (19.3 kcal/mol) is in excellent agreement with the experimental barrier (18.3 kcal/mol).38 18 ACS Paragon Plus Environment

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In the case of two-metal assisted phosphodiester hydrolysis, it is commonly proposed that a catalytic Mg(II) activates a water to act as the nucleophile with the assistance of a general base.2 Although Mg(II) is not positioned nearby the proposed catalytic base D210 in APE1, we extended our calculations to explore the possibility that a Mg(II)-ligated water can act as a nucleophile. According to our calculations, this alternative mechanism takes place through a concerted reaction (Figure S13), with a barrier (36.7 kcal/mol) that is nearly twice the value determined experimentally.38 The remarkably high calculated barrier mainly occurs due to the required change in the coordination sphere around Mg(II) from a preferred octahedral geometry49 to a pentacoordinated configuration upon cleavage of the Mg(II)-water ligation (r(Mg….Owater) = 2.39 Å in the TS), as well as the geometrically-constrained four-membered transition state (Figure S13). Moreover, the corresponding PC lies only –0.6 kcal/mol below the RC (Figure S13), which is less thermodynamically stable than the PC for the proposed two-step mechanism (–17.7 kcal/mol, Figure 5). This alternative pathway also does not explain the role of the catalytically essential D210 residue.30, 38, 40-41 Thus, the involvement of a metal-ligated water in the first chemical step seems unlikely. Overall, our predicted rate-limiting barrier and impact of key active site residues for the preferred APE1 catalytic pathway are in excellent agreement with experimental kinetic38 and mutational data.41 This gives confidence in our predicted mechanism and proposed roles for key active site residues, many of which have been disputed in the literature and/or conjectured based on crystal structures.30, 38, 40-41 Importantly, our calculations support the proposal that D210 acts as the base that initiates the reaction30 rather than neutralizing the leaving group.37 N212 plays a critical role in this first chemical step by positioning D210. Our calculations confirm previous suggestions45 that H309 neutralizes the backbone as the reaction proceeds, but constrasts proposals 19 ACS Paragon Plus Environment


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that H309 activates the nucleophilic water.37, 40 Y171 and N212 are found to perform several roles, complementing previous suggestions in the literature.30, 38, 40-41 Specifically, Y171 is located in close proximity to the backbone as previously observed in X-ray structures,38, 41 which permits the residue to help position the nucleophile at the beginning of the reaction and stabilize the substrate backbone as the reaction proceeds. Moreover, in addition to aligning D210, N212 reorients to H-bond with the substrate and provides further charge stabilization after the first step. The multiple roles played by these residues throughout the reaction are consistent with reported significant mutational impacts.41 Finally, the wealth of amino acid residues within the active site to stabilize the highly-charged intermediate permits the single Mg(II) ion to be solely involved in the second step of the reaction, activating a ligated water molecule to facilitate leaving group departure. In addition to unravelling the atomistic details of the catalytic mechanism used by a critical DNA repair enzyme to remove abasic sites from the genome, this work is a vital step toward enhancing our understanding of the ability of this enzyme to exhibit activity towards many substrates. Furthermore, since single-metal dependent nucleases share a conserved position of a metal ion,18-22 our proposed mechanism can likely be generalized to other single metal-dependent nucleases, regardless of the identity of the base to activate the nucleophile or acid to stabilize the substrate. Further studies on a range of enzymes are required to verify this proposal.

CONCLUSION In this study, a combination of different computational tools including QM, MD and QM/MM (ONIOM) methods have been used to resolve the current mechanistic ambiguity regarding the


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phosphodiester bond incision mechanism catalyzed by a prototypical single Mg(II)-dependent endonuclease, namely human APE1, which targets cytotoxic abasic sites. Detailed QM calculations identified the ligands that result in the preferred octahedral coordination sphere around the Mg(II) center, which demonstrates that ligation between the substrate backbone and Mg(II) must be avoided for enzymatically-feasible phosphodiester bond hydrolysis. Subsequent classical MD simulations coupled with the QM/MM methodology established a two-step cleavage mechanism that is initiated through nucleophilic water activation by D210 and proceeds via a phosphorane intermediate. Support for this pathway comes from the close agreement between our calculated and previously reported experimental kinetic, mutational and structural data, which permits reliable assessment of the roles of the single metal ion and key active site residues. Specifically, the critical Mg(II) cofactor is determined to facilitate the departure of the leaving group through proton transfer from a ligated water molecule. H309, as well as (albeit to lesser extents) Y171 and N212, stabilize the increased charge forming on the substrate backbone as the reaction progresses. N212 and Y171 also play key roles at the start of the reaction by positioning the D210 base and water nucleophile in the active site, respectively. Overall, our findings provide a greater understanding of the novel phosphodiester cleavage mechanism catalyzed by APE1 with the assistance of a single Mg(II) ion. These new mechanistic details will direct future investigations of the phosphodiester hydrolysis mechanisms facilitated by other single-metal dependent endonucleases, as well as aid drug design and metalloprotein engineering.

SUPPORTING INFORMATION Full Computational Details; Schematic illustration of key structural parameters along the two-step hydrolysis pathway for chemical architectures I and II, the alternative concerted mechanisms for 21 ACS Paragon Plus Environment


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architecture II, and the QM/MM model (Figures S1 and S2, S4–S6 and S9); Overlay of optimized structures for configuration a (Figure S3) or the Y171F mutant (Figure S7) and configuration c for chemical architecture II; Average geometric parameters from MD simulations (Figure S8), and overlay of the MD representative model (Figure S8) and optimized QM/MM product (Figure S10) with crystalized product complexes; QM/MM optimized structures and relative Gibbs energies for the alternative concerted hydrolysis pathway (Figure S11); comparison of structural and energetic features for select stationary points obtained with mechanical and electrostatic embedding schemes (Figure S12). Cartesian coordinates of all QM-cluster and QM/MM stationary points, as well as QM-cluster and partioned QM/MM energies.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Telephone: (403) 329-2323. Fax: (403) 329-2057. M.M.A. is currently at the Department of Chemistry, Faculty of Science, University of Damietta, New Damietta, Damietta Governorate 34511, Egypt Funding S.D.W. thanks the Natural Sciences and Engineering Research Council of Canada (NSERC; 201604568), the Canada Foundation for Innovation (22770), and the Board of Governors Research Chair program at the University of Lethbridge. ACKNOWLEDGMENT


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Computer resources were provided by NUCLEIC (New Upscale Cluster for Lethbridge to Enable Innovative Chemistry) and WestGrid, part of the Compute/Calcul Canada High Performance Computing platform. ABBREVIATIONS AP, apurinic/apyrimidinic; APE1, human AP endonuclease 1; BER, base excision repair; DFT, density functional theory; H-bond, hydrogen bond; IC, intermediate complex; QM, quantum mechanics; QM/MM, quantum mechanics-molecular mechanics; MD, molecular dynamics; NER, nucleotide excision repair; PC, product complex; RC, reactive complex; TS, transition state complex. REFERENCES 1. Schroeder, G. K.; Lad, C.; Wyman, P.; Williams, N. H.; Wolfenden, R., The Time Required for Water Attack at the Phosphorus Atom of Simple Phosphodiesters and of DNA. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4052-4055. 2. Palermo, G.; Cavalli, A.; Klein, M. L.; Alfonso-Prieto, M.; Dal Peraro, M.; De Vivo, M., Catalytic Metal Ions and Enzymatic Processing of DNA and RNA. Acc. Chem. Res. 2015, 48, 220228. 3. Bonomi, R.; Saielli, G.; Tonellato, U.; Scrimin, P.; Mancin, F., Insights on Nuclease Mechanism: The Role of Proximal Ammonium Group on Phosphate Esters Cleavage. J. Am. Chem. Soc. 2009, 131, 11278. 4. Yang, W., Nucleases: Diversity of Structure, Function and Mechanism. Q. Rev. Biophys. 2011, 44, 1-93. 5. De Vivo, M.; Dal Peraro, M.; Klein, M. L., Phosphodiester Cleavage in Ribonuclease H Occurs via an Associative Two-Metal-Aided Catalytic Mechanism. J. Am. Chem. Soc. 2008, 130, 10955-10962. 6. Cisneros, G. A.; Perera, L.; Schaaper, R. M.; Pedersen, L. C.; London, R. E.; Pedersen, L. G.; Darden, T. A., Reaction Mechanism of the ε Subunit of E. coli DNA Polymerase III: Insights into Active Site Metal Coordination and Catalytically Significant Residues. J. Am. Chem. Soc. 2009, 131, 1550-1556. 7. Casalino, L.; Palermo, G.; Rothlisberger, U.; Magistrato, A., Who Activates the Nucleophile in Ribozyme Catalysis? An Answer from the Splicing Mechanism of Group II Introns. J. Am. Chem. Soc. 2016, 138, 10374-10377. 8. Carvalho, A. T. P.; Fernandes, P. A.; Ramos, M. J., The Catalytic Mechanism of RNA Polymerase II. J. Chem. Theory Comput. 2011, 7, 1177-1188.



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Scheme 1. The general pathway characterized in this study for the APE1 catalyzed phosphodiester bond cleavage.



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Figure 1. The active site of APE1 from X-ray crystal structures of A) the Mg(II)-containing product complex (PDB ID: 4IEM) and B) the Mn(II)-containing thio-substituted substrate analogue complex (PDB ID: 5DG0).


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Figure 2. A) Schematic representation of chemical architecture I (substrate ligated to Mg(II)) and the three configurations of D70 ligation to Mg(II). B) Gibbs energy surface for each active site arrangement obtained from QM-cluster models.



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Figure 3. A) Schematic representation of chemical architecture II (substrate not ligated to Mg(II)) and the three configurations of D70 ligation to Mg(II). B) Gibbs energy surface for each active site arrangement obtained from QM-cluster models.


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Figure 4. Illustration of the entire QM/MM model (top) and the QM-layer (bottom) used in the present work. Hydrogen atoms omitted for clarity, except for the water molecules.



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Figure 5. QM/MM optimized structures (selected key distances in Å) and Gibbs energy surface for the two-step phosphodiester cleavage facilitated by APE1.


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TOC


Unveiling A Single-Metal Mediated Phosphodiester Bond Cleavage

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