Unraveling the Critical Role Played by Ado762â²OH in

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Article Ado76

Unraveling the Critical Role Played by 2#OH in the PostTransfer Editing by Archaeal Threonyl- tRNA Synthetase Mohamed M Aboelnga, John J Hayward, and James W. Gauld J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b10254 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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The Journal of Physical Chemistry

Unraveling the Critical Role Played by Ado762′OH in the Post-Transfer Editing by Archaeal ThreonyltRNA Synthetase Mohamed M. Aboelnga,1,2 John J. Hayward1 and James W. Gauld1*

1

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, N9B

3P4, Canada. 2

Department of Chemistry, Faculty of Science, Damietta University, New Damietta, Damietta

Governorate 34511, Egypt.

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ABSTRACT: Archaeal threonyl-tRNA synthetase (ThrRS) possesses an editing active site wherein tRNAThr that has been misaminoacylated with serine (i.e., Ser-tRNAThr) is hydrolytically cleaved to serine and tRNAThr. It has been suggested that the free ribose sugar hydroxyl of Ado76 of the tRNAThr (Ado762′OH) is the mechanistic base, promoting hydrolysis by orienting a nucleophilic water near the scissile Ser-tRNAThr ester bond. We have performed a computational study, involving Molecular Dynamics (MD) and hybrid ONIOM Quantum Mechanics/Molecular Mechanics (QM/MM) methods, considering all possible editing mechanisms in order to gain an understanding of the role played by

Ado762′OH

group. More specifically, a range of concerted or

stepwise mechanisms involving 4-, 6-, or 8-membered transition structures (total of 7 mechanisms) were considered. In addition, these seven mechanisms were fully optimized using three different DFT functionals namely, B3LYP, and M06-2X and M06-HF. The M06-HF functional gave the most feasible energy barriers followed by the M06-2X functional. The most favorable mechanism proceeds step-wise through two 6-membered ring transition states in which the Ado762′OH group participates, overall, as a shuttle for the proton transfer from the nucleophilic H2O to the bridging oxygen (Ado763′O) of the substrate. More specifically, the first step, which has a barrier of 25.9 kcal/mol, the

Ado762′-OH

group accepts a proton from the attacking nucleophilic

water while concomitantly transferring its proton onto the substrates C–Ocarb centre. Then, in the second step which also proceeds with a barrier of 25.9 kcal/mol, the Ado762′-OH group transfers its proton on the adjacent

Ado763′-oxygen,

cleaving the scissile Ccarb–O3′Ado76 bond, while

concomitantly accepting a proton from the previously formed C−OcarbH group.


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INTRODUCTION: The central role of the aminoacyl-tRNA synthetase (aaRS) family of enzymes is to catalyze attachment of amino acids to their corresponding tRNA.1 For each existing amino acid there is an aaRS that catalyzes its coupling onto the cognate tRNA via two half-steps within their synthetic site: activation and transfer (i.e. acylation). Specifically, within the same synthetic site, the amino acid is reacted with adenosine triphosphate (ATP) to form an aminoacyl-adenylate intermediate (aaAMP), followed by transfer of the aminoacyl (aa) moiety onto its cognate tRNAaa. Impressively, this loading process occurs with a misacylation error of ~1 in every 10,000 reactions.2 Thus, aaRSs play a key role in the accurate translation of an organism's genetic code into proteins.3 Defects in the aminoacylation process can result in misfolded and thus incorrectly functioning proteins, which can eventually lead to a variety of disease states.4 Because of structural and chemical similarities between some amino acids it can be challenging for the synthetic site of aaRSs to achieve proper discrimination. Consequently, many aaRSs exploit proofreading (editing) mechanisms that selectively act against incorrectly activated amino acids or aminoacylated tRNAaa to degrade them (Scheme 1).5 For example, several aaRSs use a tRNA-independent pre-transfer editing mechanism (Scheme 1) whereby the aminoacyl-adenylate is hydrolyzed within the synthetic site.6 However, pre-transfer editing is not always sufficient to ensure the necessary fidelity of aminoacylation, such as between isosteric amino acids. Hence, additional post-transfer editing mechanisms are often employed by aaRSs involving a second active site.7 Indeed, almost half of the aaRSs utilize post-transfer editing and thus behave as double sieve models.8 In this proofreading mechanism, misacylated tRNAaa is shuttled to the editing site where the ester bond between the incorrect aminoacyl moiety and tRNAaa is cleaved (Scheme 1). It is noted that a triple-sieve editing mechanism is used by alanyl-tRNA and prolyl-tRNA synthetases to ensure accurate aminoacylation.9



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Scheme 1. Schematic representations for the aminoacylation and editing mechanisms employed by ThrRS.

Threonyl-tRNA synthetase (ThrRS), a class II aaRS, must discriminate its substrate threonine from the non-cognate substrate serine. It is known to utilize a variety of editing mechanisms including pre-transfer editing against serinyl-adenylate.5,10-12 Unfortunately, such editing is not sufficient to achieve the necessary required high fidelity. With the exception of mitochondrial ThrRS, bacterial, eukaryotic and archaeal ThrRS all possess a remote active site for post-transfer editing.11 It is generally accepted that the editing domain sequence of ThrRS is not evolutionarily conserved.2 Indeed, distinct from bacterial and eukaryotic versions that possess a universal editing domain found in both Thr- and AlaRS, archaeal ThrRS employs a unique N-terminal post-transfer editing region.12 Accordingly, two different catalytic scenarios have been suggested for the respective editing mechanisms in bacterial/eukaryotic and archaeal ThrRS. In the posttransfer editing mechanism of E. coli (bacterial) ThrRS,12 an active site cysteinyl13 or histidyl8 residue is thought to act as the base that deprotonates the nucleophilic water and initiates the reaction. In contrast, the editing mechanism of archaeal ThrRS is thought to be a paradigm for the editing domains of most other aaRSs.14 Moreover, previous sequence analysis demonstrated a 4 ACS Paragon Plus Environment

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substantial sequence similarity between the archaeon Pyrococcus abyssi’s ThrRS (Pab-NTD) and D-aminoacyl-tRNA

deacylases (DTDs).2,15-17 The latter domain is used by aaRSs and is

responsible for hydrolyzing misacylated D-aa-tRNA, thus preserving the homochirality of proteins.18 For Pab-NTD, due to the apparent lack of direct involvement of the enzyme residues in a way that could facilitate catalysis, post-transfer editing has been suggested to take place through a substrate-assisted

mechanism.2,5

More

specifically,

based

on

experimentally

obtained

structures,8,19 the free hydroxyl group of the adenosine ribose sugar of the tRNA (Ado762′- or 3′-OH for class I and II respectively), appears to be the only potential base in close proximity to the substrate's scissile ester bond. Furthermore, a significant inhibition in editing has been observed upon its removal.8,19 Thus, it has been suggested that the

Ado762′-

or

Ado763′-OH

group of the aa-

tRNA substrate facilitates reaction by orienting a nucleophilic H2O molecule in close proximity to the aa-tRNA ester bond, i.e. the

Ado762′-/3′-OH

group plays a structural or anchoring role.8

However, the precise role played by the hydroxyl group is still debated. Post-transfer editing mechanisms in several aaRSs have been previously examined computationally. For example, Tateno and coworkers20 performed a Molecular Dynamics (MD) study in order to identify residues essential to LeuRS for editing against noncognate valine (i.e., hydrolysing Val-tRNAleu).20 A H2O molecule was observed to be consistently hydrogen bonded with the tRNAleu

Ado763′-OH

group while concomitantly positioned near the carbonyl carbon

(Ccarb) of the substrate's valinyl moiety. Quantum mechanics/molecular mechanics (QM/MM) free energy simulations on the mechanism indicated that the

Ado763′-OH

group helps activate the H2O

molecule for reaction, thus editing proceeds by substrate-assisted hydrolysis.5,14 Similarly, a QM/MM study21 on the freestanding editing domain (INS) of ProRS, which edits alanine mislinked to tRNAPro, concluded that the substrate's

Ado762′-OH

group is crucial for positioning a

nucleophilic H2O for subsequent nucleophilic attack on the substrate's Ccarb center. In this current study, we have complementarily applied both MD simulations and QM/MM methods to gain insights into the role played by the substrate's

Ado762′-OH

group in the post5

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transfer editing mechanism of Pab-NTD ThrRS. Similarities between the fully-substrate bound editing site and biocatalysts involving ribozymal catalysis have been previously noted.22,23 In particular, the function of the free RNA hydroxyl group has been suggested to either help position and anchor the nucleophilic H2O24 or to directly participate in the catalytic mechanism as in the ribosome.25 Hence, the present investigations also examined the applicability and feasibility of analogous tRNA substrate-mediated pathways in the editing site of ThrRS including concerted and step-wise anchoring, and single or double proton shuttle mechanisms (Schemes 2 and 3).

COMPUTATIONAL METHODS. Molecular Dynamics (MD) Simulations. The Molecular Operating Environment (MOE) program26 was used to prepare all chemical models for the MD simulations with the X-ray crystal structure of the editing domain of ThrRS from Pyrococcus abyssi with bound seryl-3'aminoadenosine (PDB ID: 2HL1)2 used as the initial template. The link nitrogen atom in the ester bond was replaced with an oxygen atom and the protonation states of all the residues were assigned according to the PropKa protonation tool implemented in MOE. All crystallographic water molecules were removed except for two positioned near the substrate. The model was then minimized using the molecular mechanics (MM) forcefield AMBER12. The complex was then solvated by adding a layer of water to 6 Å around the enzyme-ligand system, resulting in a system with total number of 11000 atoms. The generated chemical model was then submitted for a second MM minimization using AMBER12 until the root mean square gradient fell below 0.01 kcal/mol·Å. Using MOE26 and the NAMD program27 we submitted the resulting minimized model for unconstrained 10 ns MD simulation with a time step of 2 fs under constant pressure and temperature until the system reached equilibrium using a protocol we have successfully used in related studies.28-30 It is noted that the default settings of MOE were used which includes use of the PME method for calculating Coulombic interactions, cutoffs for non-bonded long-range interactions of between 8-10 Å, and tether ranges from 0-100 Å applied to all heavy atoms. 6 ACS Paragon Plus Environment

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Furthermore, the standard protocol that is encoded in MOE was used to parameterize any ligands using the Amber12 forcefield. The generated conformations from this MD simulation were analyzed based on their root mean square deviations (RMSD) of the heavy atoms of their active site residues (Figure S1). The obtained RMSD values were then clustered and the most representative structure (with the most prominent conformation) was chosen for the subsequent QM/MM calculations. This structure was then minimized using the AMBER12 forcefield. A suitable chemical model for the QM/MM calculations was then derived by truncating the system to only include residues and waters within 20 Å of the active site’s substrate (2000 atoms in total). QM/MM calculations: To elucidate the proofreading mechanism, we used the ONIOM QM/MM method31,32 as implemented in the Gaussian 09 program.33 This approach has been shown to be a powerful tool for examining many related catalytic mechanisms.34,35 The entire chemical model was divided into two subsystems based on their level of contribution to the reaction, Figure 1. The active region, high layer, was described using a quantum mechanical (QM) method while the remaining protein environment was treated using a MM method.

Figure 1. Illustration of the models used in this study for the molecular dynamics (MD) simulations and high-layer (QM region) of the QM/MM model.

The QM region, consisting of 88 atoms, included the substrate 3'-seryl-adenosine (Ser-Ado), a model of serine bound to the A76 residue of tRNAThr, two H2O molecules and the backbone chain 7 ACS Paragon Plus Environment


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of Pro80 and Ala82 as they are thought to stabilize the accumulated negative charge on the oxygen atom (Ocarb) in the transition state. In addition, the R-group of the invariant2 Lys121 was included as it is thought to be important in orienting and positioning the nucleophilic H2O molecule in close proximity to the substrate’s ester group.5 Glu134 was also included in the QM region due to its close proximity and previously suggested interactions with the substrate.2 To describe the QM region the density functional theory methods B3LYP,36 M06-2X and M06-HF37 in conjunction with the 6-31G(d,p) basis set were used, while the AMBER96 forcefield38 was used to describe the surrounding protein environment, i.e. the low (MM) layer. For all ONIOM QM/MM calculations the default mechanical embedding (ME) procedure was used. As noted all mechanisms were studied using the DFT functionals B3LYP, M06-2X and M06HF (see above). In particular, we evaluated the ability of these functionals to reliably and accurately describe the studied mechanisms and their thermochemistry, with the results summarized in Table 1. It was observed that the mechanism was sensitive to the % HF exchangecorrelation (%XC) included. The M06-HF functional, which has the highest contribution of the three functionals, provided a better kinetic description of the mechanism. It is also clear from this table that, M06-2X gave more reasonable energy values relative to B3LYP, in agreement with previous theoretical studies on ribozymal catalytic mechanisms.39,40 Thus, in the following discussion we will focus only on the data obtained using the M06-HF functional to describe the QM region. Hence, reported herein, optimized geometries and frequencies were obtained at the ONIOM(M06-HF/6-31G(d,p):AMBER96) level of theory, as were the corresponding Gibbs free energy corrections (ΔGcorr). Relative energies were determined by performing single point energy calculations

on

the

above

optimized

structures

at

the

ONIOM(M06-HF/6-

311+G(2df,p):AMBER96) level of theory. Only the Ca centers in the low layer were held fixed, all other atoms being free to move during optimizations.


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Table 1. Calculated energy barriers in kcal/mol for the various mechanisms (represented by their transition structure (TS) label) obtained at the ONIOM(DFT/6-311+G(2df,p):AMBER96//DFT/631G(d,p):AMBER96) + ΔGibbs level of theory. DFT Method

Free Energy of the given TS relative to the reactive complex RC I

TS4

I

TS6

I

TS8

II

TS14

II

TS24

II

TS24′

II

TS16

II

TS26

II

TS26′

B3LYP

31.1 34.8 48.5

49.3

41.9

45.6

40.3

43.1

36.2

M06-2X

34.9 33.1 38.1

39.7

36.0

39.8

30.6

29.0

40.7

M06-HF

32.7 26.1 31.5

36.9

32.3

36.0

25.9

25.9

30.5

RESULTS AND DISCUSSION: Reactive Complex: In the substrate-bound active site (RC) the active site residue Glu134 appears to play an important role in binding and positioning the Ser-Ado substrate (Figure 1). Specifically, Glu134 hydrogen bonds with both the α-amino and β-hydroxy groups of the serinyl moiety of the Ser-Ado with distances of r(Glu134COO−···+H3NαSer) = 1.57 Å and r(Glu134COO−···HOβSer) = 1.89 Å, respectively. In the average structure of the MD simulations a water molecule was observed to be consistently near to the ester bond in the substrate and concomitantly hydrogen bonded to the

Ado762′OH

group. In the QM/MM optimized structure the

oxygen of this nucleophilic water molecule (OW) was also located near the ester bond in the substrate, r(WO···Ccarb) = 2.93 Å, and with an ÐOW···Ccarb−Ocarb of 87.6°. Hence, the water appears to be well positioned for the required subsequent nucleophilic attack by OW at Ccarb. This water is held in position through the formation of a network of strong hydrogen bonds with the backbone oxygen of Pro80, r(HOH···OPro80) = 1.43 Å, the side chain ammonium of Lys121, r(OHW···NLys121) = 1.41 Å, as well as the Ado762′OH group, r(OHW···OAdo76) = 1.91 Å. Moreover, the Ocarb centre of the substrate's serinyl moiety forms a hydrogen bond with the backbone −NH− of Ala82 at a distance of 2.15 Å.



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The hydrolytic editing mechanism is initiated by nucleophilic attack of the oxygen atom of the water molecule (OW) on the Ccarb centre of the substrate, leading to the formation of a new Ccarb−OW bond. This is followed by cleavage of the ester alkoxy bond, with protonation of the bridging oxygen (Ob).

I. Concerted Mechanisms. a. Anchoring Mechanism: In this mechanism, the nucleophilic H2O molecule attacks the Ccarb atom and its proton is simultaneously shifted toward Ob atom of the ester bond through a 4-membered ring transition state (ITS4), Scheme 2. The QM/MM optimized structures are shown in Figure 2 while the corresponding free energy surfaces are shown in Figure 3.

Scheme 2. Schematic illustration of the concerted mechanisms studied: (a) the anchoring mechanism (4-membered ring); and (b) proton (6-membered ring); and (c) double-proton (8membered ring) shuttle mechanisms.


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Figure 2. The optimized structures for the reactive complex, transition structures (TSs) and product complex obtained for the concerted mechanisms with selected bond lengths in Angstroms (Å).

As can be seen in Figure 3, the free energy barrier obtained for this step is quite high at 32.7 kcal/mol and hence, this pathway is unlikely to be feasible. This high barrier may be a result of the geometrically constrained 4-membered ring. In ITS4 the hydrogen bond between the substrate's Ocarb centre and the backbone −HN− group of Ala82,

carbO···HNAla82,

has elongated

significantly to 2.30 Å, an increase of 0.15 Å from the reactive complex (Figure 2). As a result, there is less stabilization of the buildup of negative charge on the Ocarb centre. The collapse of ITS4 results in the formation of a product complex (PC1) lying 5.6 kcal/mol lower in energy than the initial reactive complex RC (Figure 3), indicating that PC1 is thermodynamically favorable. The

Ado763′O···Ccarb

Å and thus the bond is broken. Meanwhile, the

distance has now drastically elongated to 3.07

Ado762′-oxygen

of the adenosine leaving group is 11

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stabilized by the formation of a new (Ado763′O−H) single bond 0.97 Å long, while the now cleaved neutral serine moiety has a now fully formed carbC−OHW bond 1.32 Å long.

Figure 3. The calculated (see Computational Methods) Free energy (kcal/mol) surface for the concerted mechanisms to cleave the ester bond of the mischarged Ser-tRNAThr.

To better clarify the precise role for

Ado762′OH

group in the current anchoring pathway, we

investigated the mechanism in the presence of an alternate 2'-deoxy (Ado762′H) substrate (see Supporting Information). Notably, the missing hydrogen bond between the nucleophilic water molecule and the substrate’s

Ado762′OH

had only a negligible impact on the position of the water

molecule with respect to the substrate’s Ccarb. This is illustrated by an increase of just 0.07 Å in the Ccarb···OW distance compared to the corresponding distance observed in the wildtype RC. As observed in RC, the water molecule was again tightly held in this position by the formation of strong hydrogen bonds with the backbone carbonyl oxygen of Pro80 and the side chain ammonium of the conserved Lys121 residue with distances of 1.40 and 1.41 Å, respectively. 12 ACS Paragon Plus Environment

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Notably the angle of the nucleophilic attack, ÐOW···Ccarb−Ocarb, is little changed in the complex at 90.14°. Thus, the

Ado762′OH

Ado762′H

group, previously proposed8 to help position the

nucleophilic water for subsequent reaction, may not be critical towards achieving such a goal when both Pro80 and Lys121 are present. In order to fill this gap of understanding, an alternative mechanism that might provide a complete picture regarding the actual role of the

Ado762′OH

group in the mechanism has been

explored.

b.

Proton Shuttle Mechanism:

In this concerted pathway, proton transfer from the nucleophilic water to the nucleofugal

Ado763′-

oxygen atom proceeds through the Ado762′OH group (Scheme 2), i.e. the water transfers its proton to the

Ado762′-oxygen

while the

Ado762′OH

transfers its proton onto the nearby

Ado763′-oxygen.

Consequently, this pathway involves a 6-membered ring transition structure. In computational studies on ribosome-catalysed peptide synthesis, the analogous 6-membered ring mechanism was determined to proceed with a lower energy barrier relative to the corresponding 4-membered ring mechanism.41 This preference is due in part to the presence of more hydrogen bonds at more productive angles for the proton transfer processes. Indeed, the development of low-barrier hydrogen bonds is known to significantly facilitate many enzymatic reactions.42 Such interactions can form when two or more atoms with similar pKa values form strong hydrogen bonds and thus can share a proton, such as occurs within the present chemical system. For this concerted pathway, the mechanism proceeds via a 6-membered ring transition state (ITS6) with a required energy of 26.1 kcal/mol (Figure 3). This is approximately 6.7 kcal/mol lower than the value obtained for the corresponding 4-membered ring transition structure, ITS4, and is more likely to be feasible in vivo. The optimized structure of the 6-membered ring transition state (ITS6), with selected important bond lengths, is given in Figure 2. In ITS6 the developing negative charge on the substrate's Ocarb centre is again stabilized through hydrogen 13 ACS Paragon Plus Environment


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bond formation with the backbone amide −NH− of Ala82. However, this is now a much stronger interaction as indicated by its shortened length of 2.05 Å, which is 0.10 Å shorter than the corresponding distance observed in ITS4. Collapse of ITS6 leads to formation of PC1 which has been previously discussed (see above). It should be noted that we did examine other possible lower-energy concerted mechanisms that also involved a 6-membered ring transition structure. Specifically, we examined the analogous pathway using larger chemical models that might allow for better stabilization of the oxyanion centres in ITS6 by additional hydrogen bonding interactions, as has been previously suggested.43 In particular, the QM region of the chemical model was expanded to include the side chain imidazole of His83 and a second H2O molecule. This resulted in the Ocarb centre being able to form a strong hydrogen bond with the second water molecule; r(carbO…HOH) = 1.80 Å. Use of these enhanced models to examine this concerted proton-shuttling pathway resulted in only negligible changes in the reaction barrier relative to the smaller QM-region model (not shown). This suggests that additional stabilization of the charge build-up on the Ocarb atom, at least within a 4-membered ring transition structure, will not significantly lower the energy barrier obtained.

c. Double Proton Shuttle Mechanism: In the QM/MM optimized structures of RC it was observed that a second water molecule (W2), in addition to the nucleophilic water (W1) was also positioned near the experimentally critical Ado762′OH.

Therefore, we also examined possible concerted mechanisms that may involve an 8-

membered ring transition structure. Specifically, the proton transfer process from W1 to the Ado762′OH

group proceeds through W2 (i.e. a double proton shuttle mechanism), see Scheme 2

(pathway c). This pathway proceeds via the transition structure ITS8 which, importantly, lies higher in energy than RC by 5.4 kcal/mol compared to ITS6 (Figure 3 and Table 1). This increase in energy is due to the substantially larger negative entropy term (∆S‡) that is a consequence of constraining the W2 molecule within this mechanism. This negative entropy is not sufficiently 14 ACS Paragon Plus Environment

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counterbalanced by the increased number of hydrogen bonds within ITS8; the enthalpic term is only slightly lower than in ITS6 (See Table S1) because these hydrogen bonds are not at optimal distances and angles. For example, in

I

TS6 the Ocarb…HNAla82 bond distance and

ÐOcarb__Ocarb…HNAla82 angles are 2.05 Å and 156.2° respectively; in contrast, in ITS8 these optimized structural parameters now have values of 1.87 Å and 116.2°. While the length of this hydrogen bond in ITS8 is markedly shorter, it has a significantly tighter and less-linear bond angle. Consequently, proton transfer along this hydrogen bond will have a markedly higher barrier.44,45 Collapse of ITS8, like the other concerted mechanisms examined herein, leads to formation of PC1 which has been previously discussed (see above). It should be noted that, other than with the backbone −NH− group of Ala82, the three concerted-mechanism transition structures (ITS4, ITS6 and ITS8) exhibit very similar interactions between the ligand and active site residues. The most significant changes are essentially localized to the bond forming and breaking region discussed above. Notably, in ITS4 and ITS6 the bond breaking

Ado763′O−Ccarb

distances are 1.79 and 1.65 Å, respectively, while the corresponding

newly forming OW−Ccarb bonds have distances of 1.53 and 1.48 Å (Figure 2). In contrast, in ITS8 the

Ado763′O−Ccarb

and OW__Ccarb bond lengths are 1.48 and 1.61 Å, respectively; i.e. the new

OW__Ccarb bonds in ITS4 and ITS6 have essentially formed at the point of cleavage of Ado763′O−Ccarb

while the reverse is true in ITS8. This suggests that ITS4 and ITS6 are likely later

transition structures compared to ITS8.

II. Step-Wise Mechanisms: In the concerted mechanisms the nucleophilic water formally transferred one of its protons onto the

Ado763′-oxygen,

either directly or by a shuttle process. Alternatively, however, it could first

transfer a proton onto the forming oxyanion at the substrate's Ocarb centre; that is, the mechanism could proceed in a step-wise process involving the formation of a tetrahedral 1,1-diol intermediate. This may also help Lys121 and Ala82 to stabilize the negative charge built-up on the Ocarb centre during the reaction. Due in part to the previously noted geometric challenges for 15 ACS Paragon Plus Environment


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8-membered ring transition structures, we only considered step-wise mechanisms involving 4and 6-membered ring transition structures as shown in Scheme 3. Scheme 3. Schematic representation for the two-step mechanisms studied involving (a) 4- and (b) 6-membered ring proton shuttles.

a. Anchoring Mechanism: As for the above concerted pathways, we first investigated possible anchoring mechanisms, i.e. those in which the

Ado762′OH

group simply helps position the nucleophilic water (W), that

proceeds via two 4-membered ring transition structures, Scheme 3 (pathway a).25,39,46 In contrast to the concerted mechanisms, nucleophilic attack of W on the substrate's Ccarb centre now occurs with concurrent proton transfer from W onto the Ocarb centre. This step proceeds through the 4membered ring transition structure IITS14 at a cost of 36.9 kcal/mol relative to the initial reactant complex RC (Table 1). In this reaction step the proton transfer onto the Ocarb neutralizes the developing negative charge on the oxygen atom and leads to the formation of the diol intermediate complex IIIC lying 6.5 kcal/mol higher in energy than RC (Scheme 3). This low 16 ACS Paragon Plus Environment

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energy is likely to reflect enzyme-ligand and intramolecular hydrogen bonding and the neutralization of the potential oxyanion. Formation of the final product happens via one of two 4membered ring transition structures (IITS24 or IITS24′) and leads to one of two possible product complexes PC1 or PC2, respectively (see Scheme 3). In both possible reactions one the gem diol hydroxyl groups on (HO)2Ccarb transfers its proton onto the leaving Ado76O3′ oxygen. The more kinetically preferred of these two reactions proceeds through IITS24 at a cost of 32.3 kcal/mol relative to RC (Table 1). This involves the newly formed Ccarb−OWH group transferring its proton directly to the leaving

Ado763′-oxygen.

This step leads to the formation of product

complex PC2, which lies just 2.3 kcal/mol lower in energy than RC (Figure 4). Thus, while this reaction is overall exergonic, PC2 is slightly less thermodynamically preferred than PC1 by 3.3 kcal/mol. The alternative pathway proceeds via

II

TS24′ at a slightly higher barrier of 36.0

kcal/mol (Table 1) and leads to formation of PC1. These reaction high barriers (>30 kcal/mol) indicate that a two-step anchoring mechanism is kinetically disfavored and unlikely to be feasible. It has previously been noted for related systems that the charge accumulation on Ocarb is not the only factor that influences the obtained energy barriers; the constrained nature of 4-membered ring transition structure has a significant impact on the energy costs;40,47 the present results would appear to support this suggestion.

Figure 4. The calculated (see Computational Methods) Free energy (kcal/mol) surface for the most favorable step-wise mechanism occurring through a 6-membered ring transition structures. 17 ACS Paragon Plus Environment


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Figure 5. Optimized structures obtained for transition structures, intermediates, and product complex obtained for the stepwise mechanisms involving 6-membered rings.

Consequently, the feasibility of two-step mechanisms that instead proceed through 6membered ring transition structures were considered (see Scheme 3).

b. Proton Shuttle Mechanism: In these alternative mechanisms, as illustrated in Scheme 3, the initial nucleophilic attack of the water occurs with concomitant proton transfer to the

Ado762′OH

group, which simultaneously

transfers its proton to the substrate’s Ocarb atom. The formation of a high energy, unstable Ocarb oxyanion is thus avoided. This step proceeds through IITS16 with an energy barrier of 25.9 18 ACS Paragon Plus Environment

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kcal/mol (Table 1), which is slightly lower (0.2 kcal/mol) than that obtained for the corresponding concerted reaction (via ITS6), which had the lowest barrier of all concerted mechanisms (see Figure 3). As noted above, the gem diol intermediate formed, IIIC, is only 6.5 kcal/mol higher in energy than RC (Figure 4). In principle, the further reaction of IIIC to give the final product could proceed via the 4membered ring transition structures discussed above; however, those barriers were deemed unlikely to be feasible. Alternatively there are a further two different reactions possible, in which the

Ado762′OH

group facilitates cleavage of the

Ado763′O−Ccarb

bond. As for the above step-wise

anchoring mechanisms (discussed in II.a), the difference between these two pathways is the choice of which gem diol hydroxyl group acts as the proton donor onto the Ado762′-oxygen. In contrast to the 4-membered ring transition structures IITS24 and IITS24′, the kinetically and thermodynamically preferred pathway instead involves the carbC−OcarbH hydroxyl group. This step proceeds via the 6-membered transition structure IITS26 with an energy barrier equal to that of the first step in this pathway of 25.9 kcal/mol (Figure 4). This is 6.4 kcal/mol lower in energy that the lowest energy 4-membered ring mechanism (Table 1). The second possible reaction via IITS26′ occurs with a decidedly higher barrier of 30.5 kcal/mol (see, Table 1). The difference in relative energy between IITS26 and IITS26′ is also reflected in their structures (Figure 5). For example, in the lower energy transition structure IITS26 the scissile Ado763′O−Ccarb bond has elogated only slightly to a distance of 1.58 Å. In contrast, this same bond in IITS26′ is observed to be 1.87 Å indicating a later transition structure. Futhermore, in Ado762′OH···Ocarb

and

Ado762′O···HOcarb

II

TS26 the

distances are 1.32 and 1.10 Å indicating that the –OcarbH

group has almost wholly transferred its proton back onto the

Ado762′-oxygen.

Meanwhile, the

…HO3′Ado76 and Ado762′OH…O3′Ado76 interactions are almost equidistant at 1.25 and 1.20

Ado762′O

Å, respectively (Figure 5). There are also notable differences in the interactions between the transition structures and surrounding residues. Specifically, in IITS26 the partial negative charge on Ocarb is stabilized by a weak hydrogen bond to the backbone –NH– of Ala82 at distance of 2.42 Å; in IITS26′ the −OcarbH group remains neutral throughout this final step. Furthermore, in 19 ACS Paragon Plus Environment


II

TS26 the increased negative charge on the

Ado763′O

Page 20 of 28

center is stabilized by a strong hydrogen

bond with the nearby side chain ammonium of Lys121 via a bridging water molecule, r(Ado763′O···HOH) = 1.46 Å. This hydrogen bond is not observed in the higher energy transition structure IITS26′. Both product complexes PC1 and PC2 are thermodynamically favorable and lie -5.6 and -2.3 kcal/mol lower in energy than the reactive complex. The free energy surface obtained for the lowest energy pathway is shown in Figure 4. The above results suggest that in archaeal ThrRS the post-transfer editing pathway prefers to proceed through a step-wise proton shuttle mechanism involving 6-membered ring transition structures (Table 1).

CONCLUSION: Molecular dynamics (MD) simulations and ONIOM quantum mechanics/molecular mechanics (QM/MM) methodologies have been used to examine possible post-transfer editing mechanisms against Ser-tRNAThr as catalysed by the archaeal ThrRS editing site. A total of seven mechanisms, both concerted and stepwise, involving 4-, 6-, and 8-membered ring transition structures (TSs) were fully investigated to reveal the role played by the active site residues and in particular the substrate's Ado762′OH group. Mechanisms involving 4-membered ring TSs: In these mechanisms the

Ado762′OH

group aligns

the nucleophilic water molecule in close proximity to the ester group, but does not directly take part in the required proton transfers. Negligible differences from the wildtype were observed when the 2'-deoxy mutated substrate was used. The reaction barriers obtained for either concerted or the stepwise mechanisms that proceeded via 4-membered ring TSs were found to be the least kinetically favored compared to those involving 6- or 8-membered TSs. These high barriers, while having the lowest entropic cost of the three types of TSs, were the result of having the high enthalpic cost associated with such geometrically strained TSs. Mechanisms involving 8-membered ring TSs: In such mechanisms the

Ado762′OH

group helps

aligns a nucleophilic water near to the ester group and indirectly accepts a proton (via another 20 ACS Paragon Plus Environment

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active site water) from it during the reaction, and concomitantly transfers its proton onto the Ado763′-oxygen

of scissile Ccarb–3′OAdo76. The barriers for such pathways were only slight lower

than those involving 4-membered ring TSs. This was due to the fact that while being enthalpically preferred, they incurred the highest entropic costs. Mechanisms involving 6-membered ring TSs: In these TSs the

Ado762′OH

group aligns a

nucleophilic water near to the ester group and is directly involved in proton transfers onto the Ado763′-oxygen

of scissile Ccarb–3′OAdo76. Specifically, in the concerted mechanism the

Ado762′OH

group directly partakes in the proton shuttle from the nucleophilic water molecule to the

Ado763′-

oxygen. In contrast, in the two-step mechanism it first is involved in the proton shuttle from the nucleophilic water to the substrates Ocarb centre, then in the second step is directly involved in the proton shuttle from the resulting OcarbH group to the

Ado763′-oxygen.

The barriers for either

concerted (26.1 kcal/mol) or step-wise mechanisms (both reaction barriers requiring 25.9 kcal/mol) involving 6-membered ring TSs were the lowest due to the more favourable hydrogen bonding geometries as well as greater stabilization of the developing charge on Ocarb, i.e. while slightly less enthalpically preferred than the corresponding 8-membered TS's they were significantly less entropically disfavored. Importantly, in a good match with the experimental results, the Ado762′OH group is essential in such pathways to the hydrolytic correction mechanism.

Corresponding Author *Author to whom correspondence should be addressed; E-mail: [email protected]; Tel.: +1519-253-3000 Ext. 3992; Fax: +1-519-973-7098.

Author Contribution: The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Resources: 21 ACS Paragon Plus Environment


Page 22 of 28

The Natural Science and Engineering Research Council of Canada (NSERC), and M.A. acknowledges the Egyptian Cultural Affairs & Mission Sector for provision of scholarship support at the beginning of his study.

Supporting Information: The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.X Summary of the thermochemistry of the concerted mechanisms calculated at the ONIOM(M06-HF/6-31G(d,p):AMBER96) level of theory (T = 298.15 K); the Cartesian coordinates of the QM/MM optimized structures of all reactant, intermediate and product complexes, and the transition structures.

ACKNOWLEDGMENT: This work was supported by a Discovery Grant (249955-2013) funded by the Natural Science and Engineering Research Council of Canada (NSERC), with computational resources provided by Compute Canada (Project 9039). M.A. is grateful to the Egyptian cultural Affairs & Mission Sector and its Bureau in Canada (BCAC) for a graduate student fellowship provided at the beginning of his study.

ABBREVIATION: aaRS, aminoacyl-tRNA synthetases; ThrRS, threonyl-tRNA synthetases; MD, Molecular Dynamics; DFT, Density functional Theory; QM/MM, Quantum Mechanics/Molecular Mechanics; LBHB, Low barrier Hydrogen bond.

REFERENCES:


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