The T7 DNA polymerase is dependent on the host protein thioredoxin (trx) for its processivity and fidelity. Using all-atom molecular dynamics, we demonstrate the specific interactions between trx and the T7 polymerase, and show that trx docking to its binding domain on the polymerase results in a significant level of stability and exposes a series of basic residues within the domain that interact with the phosphodiester backbone of the DNA template. We also characterize the nature of interactions between the T7 DNA polymerase and its DNA template. We show that the trx-binding domain acts as an intrinsic clamp, constraining the DNA via a two-step hinge motion, and characterize the interactions necessary for this to occur. Together, these insights provide a significantly improved understanding of the interactions responsible for highly processive DNA replication by T7 polymerase. Keywords T7 DNA polymerase Molecular dynamics 2+ Introduction binding site. The presence of Mg ions via coordinate bonds with residues A476, D475, and D654, permit the formation of DNA polymerase catalyzes the synthesis of DNA—a process an octahedral coordination network with its coordination part- fundamental to the perpetuation of organisms. This is no less ners and water molecules, which pulls the reactive center of true with respect to dsDNA bacteriophages (phages). the reaction (the 3′-OH and nucleotide α phosphate) within T7 is one of the most studied model systems of phage close proximity and in the correct orientation. The interactions 2+ structure and function . The DNA polymerase of this phage of Mg with the 3′-OH of the primer, lowers the local pka and (T7Pol) is an extremely efficient DNA replication apparatus favors the formation of the reaction nucleophile. Together, this [2, 3] and was discovered in T7 infected Escherichia coli reduces the energetic costs of nucleophilic addition. The ac- mutants deficient in DNA polymerase I . T7Pol contains tive site residues K522, Y526, H506, and R518 function as a finger, palm, and thumb domain, the function of which is to hydrogen bond donors and promote optimal substrate align- position the primer-template adjacent to the nucleotide- ment for phosphoryl transfer . T7Pol is able to conduct this reaction with an extremely high level of fidelity, with error −6 −7 rates ≤2.2 × 10 for base substitutions and ≤ 3.7 × 10 and ≤ Electronic supplementary material The online version of this article −7 4.5 × 10 for +1 and − 1frameshifts,respectively . (https://doi.org/10.1007/s00894-018-3671-2) contains supplementary material, which is available to authorized users. T7Pol alone, however, is not a very processive enzyme, and requires host derived thioredoxin (trx) as a processivity This manuscript is dedicated to the memory of our esteemed colleague and friend John Quinn. factor . Stoichiometric binding (1:1) of trx to the T7Pol trx- binding domain (TBD) converts the enzyme into a highly * Damian J. Magill processive DNA replication machine, which, along with the firstname.lastname@example.org gp4 helicase, and gp2.5 ssDNA binding protein, forms the T7 replisome [8–12]. The processivity increase afforded by trx Microbial Ecology Laboratory, Microbiology, School of Natural binding is well known, with studies showing how insertion Sciences and Ryan Institute, National University of Ireland Galway, of the T7Pol and T3 polymerase TBDs in E. coli polymerase I Galway H91 TK33, Ireland and Taq polymerase, respectively, result in increased School of Biological Sciences and Institute for Global Food Security, processivity and, consequently, fidelity [13, 14]. Medical Biology Centre, Queen’s University Belfast, 97 Lisburn Interestingly, despite the redox functionality of trx, this Road, Belfast BT9 7BL, Northern Ireland 144 Page 2 of 13 J Mol Model (2018) 24:144 activity has been shown not to be necessary for the interaction Minimization, equilibration, and dynamics protocol with T7Pol [15, 16]. It has been suggested that binding of trx to the TBD results A round of steepest descent energy minimization was con- in conformational changes associated with the DNA binding ducted with an energy step size of 0.01, until a maximum −1 groove , but no analysis of the dynamics of TBD associ- potential force of <1000 kJ mol was achieved. The Verlet ation with trx has been carried out to truly determine the mo- cutoff scheme was utilized, with particle mesh Ewald (PME) tions at play and the precise functionality of trx. treatment of electrostatic interactions [23, 24]. Energy, pres- In this study, all-atom molecular dynamics (MD) simula- sure, and temperature were monitored throughout the simula- tions were utilized to determine the predominant modes of tion set up. Following on from this, NVT equilibration was motion in T7Pol and its complexes, as well as the interactions conducted for 100 ps with an integration step of 2 fs. All occurring between T7Pol, DNA, and, critically, trx. Together, bonds were constrained using the LINCS algorithm with this information was used to provide a model of trx function- holonomic constraints . PME was implemented for long- ality in the context of the T7 DNA polymerase. range electrostatics with a 9 Å cutoff for the real-space term and non-bonded Van der Waals interactions were calculated using Lennard-Jones 12-6 potentials with a 9 Å cutoff. Computational methods Temperature was maintained at 300 K by coupling to a mod- ified Berendsen thermostat . Velocities were derived Preparation of models from a Maxwell distribution. A similar set up was used for a subsequent NPT equilibration, with Parinello- The crystal structure of the T7 DNA polymerase bound to tem- Rahman pressure coupling implemented . Periodic plate DNA and the host protein trx (PDB code: 1skr) were boundary conditions were used for the systems with cutoff obtained from the protein databank . Iterative energy min- radii of 1 nm. Systems then underwent simulation for a imization was performed in Chimera followed by checking for total time of 25 ns with integration time of 2 fs and coor- steric clashes . The removal of the DNA template and trx dinates saved every 10 ps. Independent repetitions were car- allowed the creation of four structures: T7 DNA polymerase ried out to produce triplicates for each system under study (12 alone (T7Pol), T7 polymerase bound to DNA (T7Pol/DNA), simulations in total), all of which were run on the Kelvin T7 polymerase bound to trx (T7/trx), and T7 polymerase bound cluster at Queen’s University Belfast. For all properties to both DNA and trx (T7Pol/DNA/trx). All structures outlined in this manuscript, replicate graphs are provided in underwent energy minimization using the Yasara server, follow- the Supplementary Information (Figs. S1 – S16). ed by checking for clashes using Chimera and the Whatif server followed by Ramachandran analysis . In all cases, >97% of Cluster analysis and analysis of interactions residues were found to lie in favored regions and no residues were discovered in forbidden regions. Following on from this, Frames were extracted from the trajectory file every 10 ps, 2+ an Mg ion was inserted in association with the co-ordination excluding the first 2 ns of the simulation, for clustering using site of all models followed by another round of energy minimi- the Gromos method within Gromacs. A neighbourhood cutoff zation and steric clash checking in Chimera. of 3 Å was utilized for the T7Pol and a 2.5 Å cutoff for T7Pol/ DNA complexes. MD simulations Electrostatic and hydrogen bond interactions were ana- lyzed using both intrinsic Gromacs tools and the analysis fa- System set up cilities of VMD . Confirmation of interactions was carried out by distance analysis of the residues within each cluster in Hydrogen atoms were added to simulated systems according PyMol . to protonation states of individual residues with protonable side chains at physiological pH. This was driven by pKa anal- Principal component analysis ysis using PROPKA . All proteins were immersed within truncated octahedral box- Principal component analysis (PCA) was performed to isolate es of explicit solvent (TIP3P water) with a minimum clearance the major motions occurring within the concatenated systems of 20 Å between periodic images for starting configurations. via diagonalization of the covariance matrix (S) of the posi- The Amber ff99SB-ILDN force field was utilized for all simu- tional deviation of Cα atoms with respect to an average struc- lations due to its improved side chain torsion potentials. Solvent ture. This is represented as follows: + − molecules were replaced with Na and Cl ions to neutralize all charges and leave a final physiological concentration of S ¼ covðÞ r ¼ r −r T r −r T T ð1Þ ij I j IðÞ av jðÞ av 100 mM by using the genion facility of Gromacs v4.6.5 . JMol Model (2018) 24:144 Page 3 of 13 144 Fig. 1 Principal component analysis (PCA) of the dominant eigenvector and root mean square fluctuation (RMSF) analysis of T7 DNA polymerase motion. PCA of the covariance matrix of T7 DNA polymerase motions determines that the major mode of motion is localized to the thioredoxin (trx)-binding domain (TBD; highlighted in red). RMSF shows a peak of motion around the TBD, corroborating the PCA results. Note the dip in the RMSF peak, which corresponds to a backwards loop within the TBD Whereby: r represents the coordinates of the Cα atom j j r and represent the coordinates of the Cα and Cα i(av) i j S represents the element of the covariance matrix r atoms of the average structures. ij j(av) r represents the coordinates of the Cα atom T denotes the average time over the trajectory i i Fig. 2 RMSF analysis of T7 DNA polymerase. Regions corresponding to the TBD and the backwards loop region of this domain are highlighted in blue and red, respectively 144 Page 4 of 13 J Mol Model (2018) 24:144 Fig. 3 The interaction network of the backwards loop region of the T7 DNA polymerase TBD. Five networks of interaction responsible for holding together the loop are circled in varying colors, with corresponding residues colored similarly by element. Note that the loop is held by both distal and proximal hydrogen bonds in addition to the proposed F274/Y286 interaction Diagonalization of the matrix results in an orthogonal set of Affinity analysis of the T7 DNA polymerase with DNA eigenvectors revealing the directions of maximum motion within template the observed conformational space. This is presented as variance around the average structure. This analysis was carried out to see In order to provide a quantitative insight into the free en- what the predominant modes of motion were within T7Pol. ergy associated with biomolecular interactions, the Fig. 4 Root mean square deviation (RMSD) of T7 DNA polymerase over accordingly. Representative structures at these time points are displayed 25 ns all atom molecular dynamics (MD) simulation. RMSD regions showing the hinge motions of the TBD. Approximate timescales for the corresponding to observed hinge like motions of the TBD are labelled closure events are provided at their respective positions JMol Model (2018) 24:144 Page 5 of 13 144 molecular mechanics Poisson-Boltzmann surface area con- corresponds to the TBD and is perhaps unsurprisingly re- tinuum solvation (MM/PBSA) method was utilized via the vealed to be the most flexible portion of the polymerase. package g_mmpbsa . In order to make the calculations This is corroborated by the results of the root mean square tractable, trajectories were reduced to a 100 ps resolution. fluctuation (RMSF) analysis, which shows a central double Following MM/PBSA analysis, associated Python scripts peak across the range of the TBD (Fig. 2)(Fig. S1). One were utilized to derive estimates of per residue energy con- can observe a dip in this peak (highlighted by the red line) tributions to the binding process and associated plots of that is not apparent from the co-variance matrix. This dip simulation averages produced in R. occurs for a region of amino acids from 272 to 290 corre- sponding to a portion of the TBD that is looped back upon itself. This loop remains intact for the duration of the sim- ulation, showing no signs of unwinding with the overall Results and discussion movement of the TBD, accounting for the lower level of flexibility observed in the RMSF plot. The stability of this The predominant mode of motion is due to the TBD loop appearstobe mediatedinpartbya series of relatively It can be seen clearly from the co-variance matrix that there weak interactions. In this loop, a weak electrostatic contri- is a central portion of flexibility of the T7 polymerase at bution by residues E272 and K293 can be observed, but the approximately the 300 amino acid mark (Fig. 1). This major interactions maintaining the conformation of this Fig. 5 Electrostatic interactions responsible for the first hinge closure of during the first hinge closure provided in the center of the figure. Note the T7 DNA polymerase TBD. The formation of the salt bridges with that the minimal plateau of each graph (representing the establishment of time are shown in their respective graphs, and the network of interaction a stable interaction) corresponds to the time frame of the closure event 144 Page 6 of 13 J Mol Model (2018) 24:144 loop are hydrogen bonds and a putative π–π interaction The TBD closes in a two-step motion between F274 and Y286. Cluster analysis with a 3 Å neighbour cutoff yielded 11 clusters, with the primary clus- Analysis of the entire 25 ns simulation of the T7 polymer- ter being occupied for ~10.2 ns of the simulation; ~87% of ase reveals a concerted movement of the TBD. This move- all conformations occupy the first 3 clusters, and here the ment takes the form of a hinge-like action detailed in movement of residues F274 and Y286 between sandwich, Fig. 4. parallel displaced, and T-shaped conformations with a C4 The initial 5 ns reveals little in the way of overall motion (fourth carbon of the aromatic ring) atom distance mini- with respect to the TBD; however, from 5 ns to 12 ns, clus- mum and maximum of 4.6 Å and 7.8 Å, respectively, were tering of residues within the distal portion of the loop occurs observed. These distances lie above the suggested limit of and triggers the subsequent Bclosure^ of this region. The ac- 3.3–3.8 Å for π–π interactions but it is likely, considering tual closure mechanism is mediated by a series of electrostatic the flexible environment of these residues, that some inter- interactions detailed in Fig. 5 (Figs. S2 – S4). The establish- action occurs here. Hydrogen bond analysis reveals 12 ment of these interactions can be observed from ~10–15 ns, bonds occurring at an occupancy of >9.24%, which, all correlating well with the first closure event. The critical resi- together, form five distinct interaction networks holding dues involved are K290, E319, K268, E330, and R318. The this backwards loop region together (Fig. 3). The impor- K290/E319 interaction is one of those maintaining the confor- tance of this loop will become apparent later when consid- mation of the backwards loop region and likely represents a ering the role of this domain in the binding of trx/DNA. critical point of interaction. From 12 ns to 15 ns little change is Fig. 6 Electrostatic interactions responsible for the second hinge closure during the second hinge closure provided in the center of the figure. Note of the T7 DNA polymerase TBD. The formation of the salt bridges with that the minima of each graph (representing the establishment of a stable time are shown in their respective graphs, and the network of interaction interaction) corresponds to the time frame of the closure event JMol Model (2018) 24:144 Page 7 of 13 144 observed but after this a sudden movement occurs in the prox- A series of interactions surrounding the minor groove in- imal portion of the TBD that sets into motion a second closure volve major contributions with the S338 side chain bonded to event. This is again dominated by electrostatic interactions the DC9 O1P atom and the K355 side chain bonded to the (Fig. 6)(Figs. S5 – S7). A number of salt bridges are observed O1P of DT20. The latter also makes electrostatic contributions to slowly establish between residues E148/K299, K144/E314, with the DNA backbone. Lesser interactions are attributed to E149/K302, and E153/K299, reaching optimal interaction D340 bound to the DC9 O1P, similarly to its S338 neighbour, distance at the end of the simulation. These interactions likely and with R339 interacting with the O3′ atom of DT8. act to both mediate the closure event and also to lock the With respect to the major grove, the largest binding con- domain in place. tributors are R119, which predominantly binds DG4 but also Taken together, the TBD is observed to act like a clamp displays affinity for the O2P atom of DA18, and R604, which (not unlike that of the E. coli β-protein clamp), and closes in also shows dual binding to the DC3 O2P and DG2 O1P two distinct hinge-like motions, mediated by a series of elec- atoms. Other contributions are made by K404/DA5, trostatic interactions. Interestingly, closure of this domain oc- R111/DG14, and K118 to both the O1P and O2P atoms of curs in the absence of trx, which poses questions with respect DG16. Again, electrostatic interactions appear to take place to the precise role of the latter. with adjacent lysines and the DNA backbone as an additional mode of binding. Looking outside of the grooves, interactions to the rear of the DNA involving residues N436, A438, and DNA binding by T7Pol Q439 making contacts with the bases of DG4, DG22, and DG4, respectively, can be observed. The remainder of the Interactions between specific residues and DNA can be contacts are made by a significant number of residues at the crucial in determining the processivity of a polymerase terminus of the DNA helix lying within the core of the protein. and give an insight into factors underpinning polymerase Residues involved here are G442, S445, and R452. G442, fidelity. The network of interactions between T7Pol and S445, and R452 lie higher than the others, at the side below DNA is presented in Fig. 7. Fig. 7 Complete network of interaction between the T7 DNA polymerase interacting at the major and minor grooves and behind the helix are and DNA template (T7Pol/DNA). Interaction is shown at the point of colored and labelled accordingly. The catalytic triad and proposed second hinge closure with corresponding residues shown. Residues terminal stabilization triad are indicated 144 Page 8 of 13 J Mol Model (2018) 24:144 the minor groove. These interactions however, are base spe- 11 represents a T7Pol/DNA complex, whereby the TBD cific and will show differential responses depending on the remains in a completely open form, highlighting that some template sequence. form of TBD closure occurs very early in the simulation. Interestingly, three additional residues that interact in a This is most likely due to the electrostatic interactions be- base-specific manner are Y530, Q615, and E655, which lie tween positively charged residues of the TBD and the almost in a triad at the terminus of the helix. The close DNA backbone. proximity of this Btriad^ to that of the catalytic site can be observed, in particular with the adjacent residues Interactions between trx and the TBD Y530 and E655. The binding provided by these residues is specifically for the DC1 and DC24 bases, i.e., the ex- The host protein trx is critical for bacteriophage T7 viability. treme terminus. These residues may act to stabilize and Knowledge of its interactions with the TBD facilitates eluci- orientate the helix prior to its translocation to the catalytic dation of the mechanisms of fidelity in the T7 polymerase. site, which could be a crucial step with respect to the effi- Salt bridge analysis of T7 bound to trx revealed two interac- cient function of the enzyme. tions that take place between E319 and E330 of T7Pol, and Cluster analysis was utilized in order to isolate prefer- K90 and R73 of trx, respectively (Figs. 9, S8, S9). Previous ential states of the T7Pol/DNA complex across time; 11 experimental work has suggested a point of interaction be- clusters were identified within a 2.5 Å cut-off with a total tween trx and the TBD involving E319 of the TBD and G74 of 212 cluster transitions. A maximum of 51 transitions of trx . A G74D mutation abolishes binding, with com- occur between two specific clusters. Observations of clus- pensation obtained through E319K and A45T suppressor mu- ter identity with respect to time reveal that the primary tations. With respect to the present study, the importance of cluster is occupied for 50.84% of the simulation time E319 is clear, but we propose that it interacts preferentially (Fig. 8). Of all the clusters, it was found that only cluster with K90 of trx. Experimental mutation of G74 to aspartate Fig. 8 Cluster ID with respect to time for the T7Pol/DNA complex. Respective clusters are colored accordingly and displayed for the duration with which they are occupied over the course of the simulation JMol Model (2018) 24:144 Page 9 of 13 144 Fig. 9 Electrostatic interactions between trx and the TBD of T7 DNA polymerase. Progression of the salt bridge interactions are shown across a 25 ns all- atom MD simulation likely abolishes binding through a prohibitive electrostatic re- frequency bonds are observed to occur between the R73/ pulsion preventing the E330/R73 interaction. The E319K sup- V329, I72/Y265, and A93/P325 pairs, respectively, across pressor mutation likely acts via binding to the G74D mutation the duration of the simulation. It is notable that R73 engages to restore function. in a separate high occupancy hydrogen bond in addition to its In regard to other interactions, I75 of trx engages in a 79% electrostatic contribution, highlighting the importance of this occupancy hydrogen bond with T327 of T7Pol, whilst lower residue. Fig. 10 Comparison of the minimum distance of residue R288 from the DNA backbone in trx bound and unbound forms of the T7pol/DNA complex; trx bound and unbound graphs are labelled accordingly 144 Page 10 of 13 J Mol Model (2018) 24:144 Fig. 11 RMSD comparisons of T7 DNA polymerase and its complexes. Colors corresponding to each complex are provided on the plot as indicated Fig. 12 RMSF comparisons of T7 DNA Polymerase and its complexes. Colors corresponding to each complex are provided on the plot as indicated JMol Model (2018) 24:144 Page 11 of 13 144 In addition to these interactions, trx likely engages in nu- Interactions between the TBD and trx were found to con- merous hydrophobic contacts. The greatest levels of hydro- firm previous reports that trx binding reveals a number of phobicity are observed in the major helix and beta sheet re- basic residues. We found that a series of residues were ex- gion, both of which lie at the interface of the TBD. Taken posed that subsequently interact with the O1P and O2P atoms together, these form the major points of interaction between of DG10, DC11, and DG12. The most prominent of these trx and the TBD on this scale. residues are K281, K285, and R288. Analysis of hydrogen bonding between R288 and the DNA backbone reveal that bonding contributions are made in the T7Pol/DNA simula- Insights into the role of trx tion, but when one analyses the minimum distances between R288 and the DNA over time in both the T7Pol/DNA and the With respect to the actual function of trx, analysis of the crys- T7Pol/DNA/trx simulation, we find a stark difference in the tal structure of trx binding has shown that it exposes the pres- results, with a much greater level of interaction occurring in ence of a number of basic residues that are proposed to facil- the latter (Fig. 10). itate binding . This however, has never been proven from The additional electrostatic interactions afforded by trx a dynamical perspective, nor has a closed TBD conformation binding result in a more tightly bound complex with DNA, been reported. and this likely also explains the more rapid closure time of the Here, we initially found that the motion of TBD clo- TBD described above. Looking globally at the DNA interac- sure is similar in the T7Pol/DNA and T7Pol/DNA/trx tions, we also observe interactions with T271 and DG12 as simulations; however, we observe that the complete clo- well as a series of smaller contributions made by residues sure of the domain occurs at 10–11 ns, compared to ap- K268, G270, K285, K290, K300, K304, and E330. These proximately twice as long in the absence of trx. This may are characterized by low occupancy hydrogen bonds and tran- reflect a slightly distinct mode of motion from trx-free sient electrostatic interactions. Intriguingly, many of the ex- forms of the polymerase. posed basic residues lie in the backwards loop region of the Fig. 13 Molecular mechanics Poisson-Boltzmann surface area continu- graph. The per residue contributions to DNA binding are given for both um solvation (MM/PBSA) analysis of the T7 DNA Polymerase/DNA bound and unbound forms in the bottom graph. The region corresponding complex and the impact of trx binding. Total average binding energy with to residues of the trx binding domain is highlighted accordingly respect to time is given for the trx bound and unbound forms in the top 144 Page 12 of 13 J Mol Model (2018) 24:144 TBD which we observed to essentially lie along the helix. template, whilst stabilizing the most flexible portion of this This region therefore seems to be crucial in determining trx protein. This all culminates in this intrinsic clamp binding functionality with respect to T7Pol. tightly to the template, and, together with the network of in- Subsequent RMSD and RMSF analysis of the T7Pol teractions occurring between the T7 polymerase and DNA, complexes revealed intriguing differences between the forming a tightly bound complex, which is reflected in the T7Pol in all of them (Figs. 11, 12, S10–S16). Substantial high processivity and fidelity of this enzyme. This supports differences are observed in the RMSD values between single molecule fluorescence experiments that highlighted the T7Pol and its DNA- and trx-bound forms (Wilcox test, fact that trx binding suppresses microscopic hopping on and V = 2,579,900, P <2.2e−16; V = 918,610, p < 2.2e−16), off from the DNA template  respectively. The largest differences, however, were ob- The contributions made by trx are not limited to those seen served with the T7Pol/DNA/trx complex (Wilcox test, here however. It is known that interactions with the helicase V = 3,124,900, P < 2.2e-16; median difference = 0.2751 Å are also crucial in achieving high processivity . Therefore, compared with 0.0537 Å and 0.00440 Å for T7Pol/DNA additional research is necessary into the entire T7 replisome to and T7Pol/trx, respectively). This suggests that the most elucidate the full basis of fidelity. Here, however, we have stable of the three complexes is that of the T7Pol bound provided one element of this understanding. to both DNA and trx. Looking at the RMSF plot, this trend is Acknowledgment This work was supported by the Science Foundation the same, but, additionally, we observe the localization of this Ireland. proposed stability to the TBD as shown by lowering of the double peak region corresponding to this domain. Open Access This article is distributed under the terms of the Creative Interestingly, the stability provided by DNA is negligible, Commons Attribution 4.0 International License (http:// but the presence of DNA is nevertheless necessary to achieve creativecommons.org/licenses/by/4.0/), which permits unrestricted use, full stability due to trx binding. This is likely due to the in- distribution, and reproduction in any medium, provided you give creased DNA interactions facilitated by the exposed residues. appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. MM/PBSA analysis of T7 DNA polymerase interactions with the DNA template References In order to provide a quantitative insight into the binding 1. Demerec M, Fano U (1945) Bacteriophage-resistant mutants in of T7Pol for the DNA template and the impact of trx Escherichia coli. 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Journal of Molecular Modeling – Springer Journals
Published: Jun 1, 2018
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