Molecular Simulations on the Thermal Stabilization of DNA by

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Molecular Simulations on the Thermal Stabilization of DNA by Hyperthermophilic Chromatin Protein Sac7d, and Associated Conformational Transitions U. Deva Priyakumar,* G. Harika, and G. Suresh Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500 032, India ReceiVed: February 22, 2010; ReVised Manuscript ReceiVed: October 12, 2010

Sac7d belongs to a family of chromosomal proteins, which are crucial for thermal stabilization of DNA at higher growth temperatures. It is capable of binding DNA nonspecifically, and is responsible for the increase in the melting temperature of DNA in the bound form up to 85 °C. Molecular dynamics (MD) simulations were performed at different temperatures on two protein-DNA complexes of Sac7d. Various structural and energetic parameters were calculated to examine the DNA stability and to investigate the conformational changes in DNA and the protein-DNA interactions. Room temperature simulations indicated very good agreement with the experimental structures. The protein structure is nearly unchanged at both 300 and 360 K, and only up to five base pairs of the DNA are stabilized by Sac7d at 360 K. However, the MD simulations on DNA alone systems show that they lose their helical structures at 360 K further supporting the role of Sac7d in stabilizing the oligomers. At higher temperatures (420 and 480 K), DNA undergoes denaturation in the presence and the absence of the protein. The DNA molecules were found to undergo B- to A-form transitions consistent with experimental studies, and the extent of these transitions are examined in detail. The extent of sampling B- and A-form regions was found to show temperature and sequence dependence. Multiple MD simulations yielded similar results validating the proposed model. Interaction energy calculations corresponding to protein-DNA binding indicates major contribution due to DNA backbone, explaining the nonspecific interactions of Sac7d. 1. Introduction Highly abundant DNA binding chromosomal proteins of certain hyperthermophilic archaea play a vital role in DNA stabilization at high growth temperatures and compaction in absence of histones.1,2 Sulfolobus acidocaldarius is a hyperthermophilic organism which exhibits an optimum growth temperature around 85 °C.3-5 A 7 kDa protein from S. acidocaldarius, Sac7d, is capable of binding DNA nonspecifically, thereby increasing its thermal stability.6 Previous studies have shown that the melting temperatures of DNA duplexes increase to about 85 °C upon binding to Sac7d achieved via protein-DNA interactions.6,7 Hyperthermophilic proteins such as Sac7d are of great interest and are excellent model systems for understanding the factors responsible for protein stability in general. These proteins also have potential applications in biocatalysis, since the reactions catalyzed by hyperthermophilic enzymes are feasible at high temperatures that may possibly help in enhancing the yield and/or rate.8-11 While the stabilization of DNA by Sac7d at elevated temperatures has been shown by various studies, the atomic details of such a remarkable effect is not clearly understood. Sac7d binds to the minor groove of DNA nonspecifically causing a single step kink of ∼60° with a protein-DNA interface extending to the length of about four base pairs.4 Structures of several protein-DNA complexes of Sac7d have been characterized using X-ray crystallography.4,12-14 Comparison of these structures with the available protein structures reveal minimal differences in the three-dimensional structure of the protein upon DNA binding.4 In contrast, DNA undergoes * To whom correspondence should be addressed. Phone: +91-40-6653 1161. Fax: +91-40-6653 1413. E-mail: [email protected].

significant structural changes with respect to their backbone conformations in addition to the single step kink pointing to an induced fit mechanism for binding. Sso7d, a homologue of Sac7d, found in Sulfolobus solfataricus has been shown to exhibit similar DNA binding properties.14-16 DNA bending induced by intercalation of V26, and M29 along the minor groove has been recognized as crucial for the protein-DNA binding.7,12 Robinson et al. were the first to obtain the crystal structures of Sac7d bound to DNA that revealed the protein-DNA interface, and the DNA kinking.4 Wang and co-workers have characterized several X-ray crystal structures involving mutants of Sac7d, and with different sequences of DNA including in presence of a mismatched base pair.12-14 They have also obtained the structure of an engineered DNA binding hyperthermophilic protein with Sac7d and GCN4 as templates.17 A model structure for a multimeric Sac7d-DNA complex has been obtained using small-angle X-ray scattering study, along with molecular modeling techniques.18 While several studies reported static structures of DNA complexes of Sac7d, a molecular level understanding of how the protein is able to stabilize DNA molecules, and their dynamics is lacking. However, it should be noted that experimental studies have provided vital information on the importance of certain residues in protein stability, and DNA binding activity.19 Edmonson, Shriver, and co-workers have extensively investigated the thermodynamics of DNA binding.20 In one of the studies, they have shown the importance of the only tryptophan residue, which forms a single hydrogen bond with a base of the DNA.19 Mutations of W24 were found to significantly diminish the DNA binding activity of Sac7d. Similarly, they have also investigated the effect of V26 and

10.1021/jp101583d  2010 American Chemical Society Published on Web 11/18/2010

Thermal Stabilization of DNA

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Figure 1. (a) Binary complex of the Sac7d protein bound to a DNA (PDB ID: 1AZP). The protein is given in the cartoon representation and colored green, and the intercalating residues V26 and M29 are given in the sphere representation. The kinking site in the DNA is marked by a red ellipse. (b) The two DNA sequences, AZP and AZQ studied here; the intercalation site in each of these sequences is marked by a bold line.

M29 that are involved in intercalation of the DNA on the thermodynamics of DNA binding using several mutants.7 Using fluorescence and circular dichroism spectroscopic studies, McAfee et al. showed that Sac7d strongly binds to GC-rich sequences compared to AT rich sequences, though the actual binding is sequence-independent.20 The temperature dependence of the free energy and enthalpy of binding was examined by Peters et al.6 The binding affinity was found to increase with temperature, and the enthalpy of binding was found to be positive, which decreased linearly with temperature. The unfavorable enthalpy of DNA binding was suggested to be related to the energy expense toward bending the DNA.6,7 In addition to the sharp kink in the DNA structures upon binding to Sac7d, B- to A-form transitions have been observed.21 The extent of these transition, and whether they occur with respect to all the deoxyribose moieties in the DNA or only in select few of them are not very clear. In the present work, an attempt is made to understand the atomistic details of the structural transitions occurring in the DNA, the role of Sac7d in facilitating the stabilization of DNA duplexes, and their temperature dependency. Molecular dynamics (MD) simulations are valuable tools for investigating biological systems and processes at an atomistic level.22 The structural and energetic determinants of the unusual thermal stability of hyperthermophilic proteins have been reported.23-26 de Bakker et al. and we have investigated the structure and stability of Sac7d protein using MD simulations.23,26 In the present work, we report results from MD simulations on binary complexes of Sac7d with two different DNA sequences (Figure 1)sd(GCGATCGC)2 and d(GTAATTAC)2 referred to as AZP and AZQ in the remainder of the manuscriptsperformed at different temperatures. In addition to the sequence differences in the two DNA molecules, Sac7d is bound to d(GCGATCGC)2 with a sharp kink at the C2G3 step and at A3A4 step in d(GTAATTAC)2.4

Initial structures for the protein-DNA complexes were taken from previous studies (PDB IDs: 1AZP and 1AZQ).4 All of the MD simulations and the analysis of trajectories were done using the CHARMM biomolecular simulation program.27 The CHARMM22 all atom protein force field along with the CMAP corrections, and CHARMM27 all atom nucleic acid force field parameters were used.28-31 A modified TIP3P model was used for the explicit solvent environment in all of the MD simulations reported here.32 The missing hydrogen atoms were added to the coordinates obtained from PDB using the HBUILD option in CHARMM. The resulting structures were overlaid on to a truncated octahedron water box whose size was chosen so that the distances between the edge of the box and the protein were about 9 Å in all directions. Nonphysical contacts of the protein-DNA complexes with the water molecules were eliminated by deleting the solvent molecules whose oxygen atoms were present within 2.2 Å of any of the non-hydrogen atom of the solute. Eight sodium ions were placed at random positions to make the water box electrically neutral. In the following energy minimizations and MD simulations, the Lennard-Jones (LJ) interactions and the calculation of real space electrostatic interactions were truncated at 10 Å, and a force switch smoothing function was used from 8 to 10 Å. The particle mesh Ewald summation methodology was used for calculating long-range interactions.33 Periodic boundary conditions, imposed by CRYSTAL module, were used in all the calculations.34 The solvated protein-DNA complexes were subjected to a 500-step adopted basis Newton-Raphson (ABNR) minimization, and they were equilibrated for 100 ps using MD simulations performed at 300 K in the NPT ensemble. Mass weighted harmonic constraint with a force constant of 5 kcal/ mol/Å2 was applied to all of the non-hydrogen atoms of the protein and DNA during these minimizations and equilibrium simulations. After removing the harmonic constraints, the systems underwent 500-step ABNR minimizations, and production simulations were run for 15 ns. For the simulations at 360 K, the systems were gradually heated from 300 to 360 K during a 20 ps MD simulations. This was followed by equilibration and production simulations for 15 ns at 360 K, as described above. For both AZP and AZQ, two independent MD simulations were done at 360 K starting from different sets of initial velocities. These simulations will be referred to as 360-1 and 360-2 in the remainder of the paper. Similarly, MD simulations were carried out at 420 and 480 K on both the protein-DNA complexes. To compare the behavior of the DNA in the absence of the protein, MD simulations were performed at all four temperatures (300, 360, 420, and 480 K) using a similar protocol presented in this section. SHAKE algorithm35 was used to constrain the covalent bonds involving hydrogen atoms in all the energy minimizations and MD simulations. Nose-Hoover thermostat36 and Langevin piston algorithm37 were used to achieve the NPT ensemble. For a study of this kind, it is desirable to use replica exchange MD simulations38 that would enable adequate sampling at each of the temperatures. However, it is not practical due to the computational complexity considering the size of the systems considered here. The calculations performed here are expected to be adequate enough to obtain qualitative trends of the temperature effects on the structure and dynamics of the protein-DNA complexes. Moreover, the agreement between the results obtained from two different simulations performed at 360 K on the protein-DNA validates such a protocol used in the current study.

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TABLE 1: Average RMSD Values (Å) of the Protein-DNA Binary Complex, Protein and the DNA Structures with Respect to the X-ray Crystal Structures, and Canonical A- and B-Forms AZP

300

360-1

360-2

420

480

Protein-DNA Protein DNA (Complexed) - xtal DNA (Complexed) - B form DNA (Complexed) - A form DNA (Alone) - xtal DNA (Alone) - B form DNA (Alone) - A form

1.21 ( 0.04 1.03 ( 0.01 1.13 ( 0.04 2.72 ( 0.06 2.29 ( 0.04 2.01 ( 0.01 1.39 ( 0.02 2.52 ( 0.01

1.77 ( 0.08 1.41 ( 0.08 1.43 ( 0.06 2.58 ( 0.11 2.59 ( 0.03 3.11 ( 0.61 3.06 ( 0.76 3.69 ( 0.61

1.81 ( 0.26 1.20 ( 0.04 1.73 ( 0.32 2.91 ( 0.15 2.74 ( 0.23

6.73 ( 1.65 1.95 ( 0.19 5.86 ( 1.40 6.50 ( 1.30 6.20 ( 1.32 8.21 ( 1.43 8.57 ( 1.46 8.16 ( 1.41

9.52 ( 0.58 2.59 ( 0.33 9.32 ( 0.40 9.44 ( 0.26 9.03 ( 0.31 12.93 ( 0.33 13.34 ( 0.33 12.82 ( 0.32

AZQ Protein-DNA Protein DNA (Complexed) - xtal DNA (Complexed) - B form DNA (Complexed) - A form DNA (Alone) - xtal DNA (Alone) - B form DNA (Alone) - A form

300 1.72 ( 0.05 1.32 ( 0.03 1.36 ( 0.02 3.46 ( 0.02 2.58 ( 0.01 2.65 ( 0.04 1.56 ( 0.03 2.42 ( 0.04

360-1 2.30 ( 0.08 1.35 ( 0.11 2.21 ( 0.08 4.11 ( 0.04 3.06 ( 0.03 10.12 ( 2.54 10.75 ( 2.67 10.07 ( 2.59

360-2 2.52 ( 0.21 1.26 ( 0.09 2.48 ( 0.27 3.72 ( 0.21 3.12 ( 0.17

420 12.50 ( 0.96 1.29 ( 0.11 12.53 ( 0.67 13.46 ( 0.55 12.67 ( 0.62 12.09 ( 0.55 12.59 ( 0.52 12.03 ( 0.49

480 10.39 ( 0.39 3.34 ( 0.30 10.89 ( 0.44 11.59 ( 0.40 11.00 ( 0.40 12.39 ( 0.79 12.97 ( 0.77 12.26 ( 0.81

Structural coordinates were saved every 2 ps during the production simulations, and were used for the analysis. VMD39 was used for visualization purposes, and for depicting structural representations. Interaction energy calculations were done using the INTER command implemented in the CHARMM program. Intrastrand stacking interaction was calculated as the interaction energy between a given base, and its two adjacent bases each present on the 5′ and 3′ sides of the same strand. Interstrand stacking interaction was calculated as the interaction energy between the base with adjacent bases that are present on the 5′ and 3′ sides of the complementary strand. For example, the intrastrand interaction energy of G3 is its interaction with C2 and A4 of the same strand, and its interstrand interaction energy is calculated as the interaction of G3 with G7 and T5 from the complementary strand. Estimation of root-mean-square fluctuations (RMSF) from experimental temperature factors was done using the following equation. The data presented in the tables and figures were obtained from the last 12 ns of the MD simulations unless otherwise mentioned. The errors presented in the tables represent the standard error of the mean calculated from block averages obtained at every 3 ns window during the last 12 ns of the simulations. The kink in the DNA was estimated using the “roll” helicoidal parameter40 calculated using the Curves+ program.41

8 β factor ) π2*(RMSF)2 3 3. Results and Discussion The structures of the DNA molecules in the protein complexes were investigated based on base-pair distances, backbone conformational preferences, roll angle related to the kink, solvent accessibility, and deviations from the reference structure. The proteins-DNA interactions were examined using interaction energies within the context of the binary complexes, and interatomic distances involving select protein-DNA contacts. The structure, dynamics, and stability of DNA are presented first followed by the protein-DNA interactions. 3.1. Structure and Dynamics. Initially, the structures obtained using the MD simulations were compared to the X-ray crystal structures. The root-mean-square deviations (rmsd) of the protein-DNA binary complexes obtained at 300 K with respect to the reference structures are 1.2 and 1.7 Å, indicating good agreement (Table 1). The time series of the rmsd values

are given as a Figure in the Supporting Information (Figure S1). The rmsd values calculated from the 360 K simulations exhibit marginally larger deviations with values of 1.8 Å, and 2.3 and 2.5 Å for AZP and AZQ respectively. Such rmsd values of 2.5 Å or less indicate good agreement with the reference structure, and show that the binary complexes are stable at temperatures up to 360 K. The structures display substantial deviations from the experimental structure at the other two higher temperatures (420 and 480 K). Previous experimental and computational studies have shown that the Sac7d protein is quite stable at temperatures around 360 K.19,23,42,43 In order to evaluate the structural deviation of the protein structures, rmsd values were calculated only on the CR atoms of the protein. In all of the six MD simulations performed at 300 and 360 K, the structural deviations are minimal, exhibiting values from 1.0 to 1.4 Å. This observation further substantiates the previous studies that Sac7d retains its three-dimensional structure even at 360 K. More importantly, the presence of DNA does not seem to influence the structure of protein at both the temperatures. Interestingly, the protein did not completely denature even at 420 and 480 K within the time scale of the simulations. In our previous study, Sac7d was found to denature within the same simulation time at 500 K in the absence of the DNA. Similar calculations on the DNA within the binary complexes show that the DNA molecules exhibit larger deviations than the proteins in general. However, the average rmsd values of about 2.5 Å or less at 300 and 360 K are within the acceptable range observed in a typical nucleic acid simulation, indicating that they are quite similar to the X-ray structures. The table also presents the rmsd of the bound DNA with respect to the canonical A- and B- forms of the same sequence. The values of deviations indicate that the DNA is close to the A-form than to the canonical B-form when complexed to the protein. However, both of the values are reasonably larger than the values obtained with respect to the X-ray crystal structure, which is likely due to the kink in the structures. Calculations of pseudorotation angles of the sugar moieties reveal that the DNA exhibits both A- and B-like in the local regions depending on the binding pattern (see below). DNA alone simulations show that they are close to canonical B-form at 300 K as expected, and denature at all other temperatures. The extent of the deformation of the DNA structures, and the ability of Sac7d in stabilizing the bound DNA molecule are discussed below. Contact maps that represent the inter-residue/nucleotide dis-


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Figure 2. Root mean square fluctuations of the residues of the protein (a and b), and the nucleotides of the DNA (c and d) obtained using the last 12 ns of the different MD simulations. RMSF values obtained for the protein only systems at 300 and 360 K are also given for comparison. Fluctuation data obtained from the temperature factors of the protein residues are shown in black. The β-strands and the R-helix are represented by gray block arrows and block, respectively.

tances are capable of illustrating inter and intramolecular interactions in the protein-DNA complexes (Figures S2 and S3 in Supporting Information). The contact maps corresponding to protein, DNA, and the protein-DNA interface obtained at 300 and 360 K are similar for the both the binary complexes indicating retention of the structural features observed in the experimental structure. Additionally, comparison of the contact maps reveal that the intramolecular interactions of the protein within the binary complexes obtained at 300 and 360 K are similar to those obtained for protein only systems.23 In the simulations done at 420 and 480 K, the base pairing in the DNA, and the protein-DNA contacts are diminished, showing their instability. As discussed above, the structure of protein itself in the binary complex is less affected during the two high temperature (420 and 480 K) simulations. The dynamic features of the binary complexes were investigated based on calculation of root-mean-square fluctuations (RMSF) with respect to the average structure during the last 12 ns of the simulations (Figure 2). The RMSF values obtained for Sac7d at 300 and 360 K reported in our previous study are also included for comparison.23 Data from the 420 and 480 K simulations are not provided since the increased fluctuations at these temperatures are not relevant. The flexibility patterns of the protein residues in the protein-DNA complexes were observed to be similar to those obtained for the protein only systems. The RMSF values calculated at 360 K are marginally higher than 300 K; however, the flexibility patterns are similar confirming minor changes in the structure of the protein. The figure also depicts the RMSF values calculated from the temperature factors that were obtained from the X-ray crystallography data.4 A rigorous comparison between the observed and calculated fluctuation measurements is not possible since they were calculated in the crystal environment and from MD simulations in explicit solvent environment, respectively. Nonetheless, qualitative agreement is seen between these two data (Figure 2).

However, the nucleotides of the DNA molecules exhibit flexibility patterns that are significantly different at two different temperatures. Understandably, the terminal nucleotides along each of the 5′ and 3′ ends are found to be more flexible compared to the central nucleotides as the terminal base pairs in general are prone to base opening and fraying.44 However, in the current simulations, two terminal base pairs along each of the ends of the duplexes are found to sample a larger conformational space especially in the AZQDNA at 360 K. Further investigation of the structure of the DNA reveals that these base pairs undergo base pair opening, since the protein is able to stabilize only four to five base pairs, leading to higher RMSF values (see below). 3.2. StructureandStabilityoftheDNAMolecules.3.2.1. Structural DeWiations. The preceding section showed that the structures of the DNA molecules have undergone larger structural changes at 360 K compared to 300 K with respect to the X-ray crystal structures. However, visual inspection of the structures from the MD simulations indicates that only part of the DNA, especially one or two terminal nucleotides, deviate from the starting structure. Hence, the extent of deformation of the DNA structures at 360 K was evaluated by calculating the rmsd values for nucleotides involving every 2, 3, 4, and 5 base pairs (Table S1 in the Supporting Information). In AZP, all of the base pair steps exhibit rmsd values of about 1.5 Å or less. Similar calculations on the 360 K simulations of AZQ indicate larger deviations in T2A7 base pair in addition to the terminal base pairs. Such a structural deviation of T2A7 is observed consistently in both the three MD simulations performed at 360 K. Visual inspection of the X-ray crystal structures of the two protein-DNA complexes indicate that the kinking sites are at the C2G7-G3C6, and A3T6-A4T5 base pair steps in AZP and AZQ, respectively (Figure 1). This reveals a model where Sac7d facilitates stabilization of the three-dimensional structure of DNA up

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Figure 3. Probability distributions corresponding to the N1(G/A)N3(C/T) distances of the central base pairs of the DNA in AZP (a), and AZQ (b). Data obtained at 300 K are given in black, and those obtained at 360 are given in red and blue.

Figure 4. Probability distributions of the pseudorotation angle corresponding to the puckering of the deoxyribose moiety of the DNA. The regions corresponding to C3′-endo, C2′-endo, and O2′-endo are marked appropriately. Data obtained at 300 K are given in black, and those obtained at 360 are given in red and blue.

to five base pair steps, where the kinking of the duplex occurs in the first two base pair step. This hypothesis is further validated based on other structural and energetic analysis as discussed in the following sections. 3.2.2. Base Pairing and Stacking. Base pair interactions in the DNA structures were examined based on the probability distributions corresponding to the N1(GUA/ADE)-N3(CYT/ THY) interatomic distances of the central six base pairs of the DNA in the protein-DNA complexes (Figure 3, and Figures S4-S7 in the Supporting Information). A distance of about 3 Å corresponds to Watson-Crick (WC) type base pairing, and values of more than 5 Å indicate disruption of base pair interactions. In addition, the percentage of base closed states were calculated based on two different geometric criteria: first, a base pair is assumed to be open if the solvent accessible surface area (SASA) of the imino proton and the nitrogen covalently attached to it (N1-H1 of GUA and N3-H3 of THY) is nonzero, and closed if the quantity is zero (Table 2); second, based on the N1-N3 distance with a cutoff of 3.5 Å (Table S2

in Supporting Information). Both of the sequences of the DNA (AZP and AZQ) maintain base pairing characterized by sharp peaks at 3 Å at 300 K. However, at 360 K, nonzero probabilities are observed for certain base pair distances more than 5 Å indicating the sampling of partially base open states especially in AZQ. The five base pairs from the kinked site of the DNA (C2G7 to C6G3) of AZP were observed to be stable, and peaks corresponding to ∼3 Å are observed. In contrast, A3T6 to A7T2 of AZQ are found to undergo partial base opening at 360 K exhibiting nonzero probabilities beyond 5 Å. Notably, T2A7 located one base pair above the kinked site, samples regions corresponding to base open states considerably in both the simulations further substantiating the hypothesis that Sac7d is able to stabilize only up to five base pairs including the ones that are part of the kinked site. The percentages of base closed states calculated based on both the measures given above support the stability of the DNA complexed to the protein at 360 K. However, the DNA alone systems denature at his temperature exhibited by larger values of the percentages. In the AZP DNA alone system, two base pairs (A4T5 and T5A4) remain in the

TABLE 2: Percentage of Base Pair Closed States Based on Solvent Accessible Surface Area (Å2) of the Imino Proton and the Nitrogen Covalently Bound to It (N1 and H1 of Guanine, and N3 and H3 of Thymine) DNA (Alone)

DNA (Complexed)

AZP

300K

360K

-420K

480K

300K

360K-1

360K-2

420K

480K

C2-G7 G3-C6 A4-T5 T5-A4 C6-G3 G7-C2

99.9 100.0 100.0 100.0 100.0 100.0

43.8 77.8 99.1 99.7 83.3 87.5

45.0 63.2 36.8 34.4 32.0 24.9

21.9 11.4 8.2 8.3 9.6 37.6

41.2 45.9 100.0 100.0 100.0 100.0

62.2 73.2 99.9 100.0 99.9 94.6

32.7 72.1 100.0 100.0 100.0 78.9

23.2 64.9 50.8 59.8 66.5 47.6

33.7 21.0 16.3 30.3 21.4 13.4

AZQ T2-A7 A3-T6 A4-T5 T5-A4 T6-A3 A7-T2

300K 99.8 100.0 100.0 100.0 100.0 100.0

360K 3.5 39.5 48.7 47.0 44.1 17.7

420K 9.5 7.9 19.2 22.8 14.6 3.6

480K 3.6 3.7 1.3 5.5 4.7 1.8

300K 99.9 57.4 72.9 100.0 100.0 100.0

360K-1 40.8 80.6 78.6 99.8 92.2 49.0

360K-2 57.2 16.7 95.8 99.9 99.9 73.9

420K 3.8 8.1 6.5 13.2 27.3 11.8

480K 5.7 5.1 11.8 8.4 12.0 3.2

A given base pair is classified as base pair closed if the defined atoms are not accessible to the solvent, and as open state if the atoms are accessible to the solvent.



Figure 5. The probability distributions of the pseudorotation angles of the sugar moieties of each of the ten nucleotides shown below. The green spikes represent the pseudorotation angle calculated from all the available experimental structures.

closed state at the end of the 15 ns simulation. The energetic contribution of the base pair interactions were obtained by calculating the interaction energies between the individual bases forming each of the central base pairs of the duplexes (Table 3). The interaction energies between G and C, and A and T in isolated GC and AT base pairs based on geometry optimized dimers using CHARMM force field are about -22.5 and -11.0 kcal/mol, respectively.28 The interaction energies of base pairs C2G7 to C6G3 in AZP obtained from all simulations are comparable to the energies given above indicating high stability. The central three base pairs (A4T5 to T6A3) in AZQ are found to be more stable exhibiting interaction energy values similar to optimized dimers. The lowering of the favorable interaction energies corresponding to the A3T6 (in 360-2), and A7T2 (in

360-1) in one of the simulations are due to the partial base opening events (Figure 3). Stacking interactions between adjacent bases are crucial for the stability of nucleic acids in general, and for maintaining their helical structure.45 The intra- and interstrand interaction energies of each of the four central base pairs were calculated to examine the effect of temperature on the stability of DNA (Tables S3 and S4 in the Supporting Information). Stacking interactions involving guanine bases are more favorable than that of the other bases consistent with previous reports.46 The stacking interactions calculated from the 360 K MD simulations are comparable with respect to the values obtained from the room temperature simulations indicating that the stacking phenomenon is mostly unaffected. The DNA sequences studied

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here are palindromic, and hence the bases in similar positions in the two strands are expected to have similar stacking effects. However, the differences in the stacking interactions involving purine bases are traced to the disruption of stacking due to kinking of the DNA by Sac7d. For example, the intrastrand stacking interactions of 1A3 and 1A4 with the adjacent bases of AZQ are less favorable compared to 2A3 and 2A4 in all of the three simulations because of the kinking at the A3A4 step. Such a disruption of intrastrand stacking interaction energies is not observed in AZP since the kinking does not involve two purine bases (C2G3). But, similar effect is exhibited in the interstrand interactions of G3 in AZP, where stacking interactions of 1G3 are significantly less favorable compared to 2G3. In DNA alone, simulations at 300 K, the inter and intra strand stacking interaction energies reflect the symmetry in the DNA sequence in the absence of the kink. Overall, the base pair interaction energies and stacking interactions calculated from simulations done at 300 and 360 K are similar supporting the stabilization of DNA by Sac7d at elevated temperatures. In the above sections, we have shown that the DNAs are stabilized at temperatures only up to 360 K, and undergo denaturation at 420 and 480 K. Hence, other analyses involving the conformational transitions, and protein-DNA interactions concentrated only on the trajectories obtained at 300 and 360 K. 3.2.3. B- to A-Form Transitions in DNA. DNA duplexes exist primarily in a B-form conformation, which is characterized by certain values of the phosphodiester backbone torsion angles and pseudorotation angles of the deoxyribose moieties. DNA has been shown to undergo structural changes in order to accommodate itself in the binding pocket, one of them being the kinking and bending related transitions. Circular dichroism studies reported by Edmonson, Shriver, and co-workers have shown that DNA undergoes structural transitions with respect to their backbone conformational preferences upon binding to Sac7d.20 Later, Welfle and co-workers characterized these structural changes as B- to A-form transitions using Raman spectroscopy.21 Additionally, the X-ray crystal structures showed the existence of certain nucleotides in the north conformation.4,12-14 However, the extent of these transitions in each of the nucleotides, and their temperature dependence is not known. The effect of protein binding, and elevated temperatures on the conformational preferences of the two DNAs considered here was investigated by calculating the probability distributions corresponding sugar pseudorotation angles (Figure 4). The rmsd calculations reveal that the DNA structures within the binary complexes are close to canonical A-form than the B-form. However, the kink region dominated the large deviation with respect to both the forms. Here, we show that the local regions of the DNAs sample A- or B- form regions depending on the location of binding. These are attributed to the hydrophobic residues that facilitate such a B- to A- form transition in the DNA (see below). The DNA molecules sample regions corresponding to the sugar pseudorotation angles of C2′-endo, O4′-endo, and C3′-endo conformations, which is in good agreement with experimental studies.12,14 The analysis also reveals that the north conformation (C3′-endo) is sampled more at 360 K compared to at 300 K in both AZP and AZQ. An opposite trend is seen with the sampling of O4′-endo form, with the DNA molecules sampling more at 300 K with respect to 360 K. The increase or decrease in the probabilities corresponding to C3′endo or O4′-endo conformation respectively while going from 300 to 360 K is more dramatic in the AT rich sequence (AZQ). It is quite apparent that the enhanced sampling of sugar pseudorotation angles corresponding to C3′-endo and O4′-endo

Priyakumar et al.

Figure 6. Probability distributions of the roll angle calculated at the intercalation site calculated for the DNA in the two protein-DNA complexes at 300 and 360 K, and for DNA alone system at 300 K. The data has been plotted from 300 to 0° followed by 0° to 300° for better visualization of the data.

conformations is due to the protein-DNA complex formation. To better understand which nucleotides undergo structural transitions, and to what extent, probability distributions were calculated for each of the deoxyribose ring pseudorotation angles and compared with the structural data from all the existing experimental structures (Figure 5). The plots are arranged with respect to the DNA kink site (see Figure 5 and caption for more details). The probability distributions calculated for similarly positioned nucleotides are comparable for the two sequences, and with the available experimental pseudorotation angles calculated from all the X-ray crystal structures. Nucleotides vicinal to the intercalation site (N1a-N1b to N3a-N3b) exhibit interesting conformational behavior. In contrast, N4/N5 which are located farther from the kinking site sample mostly conformations corresponding to C2′-endo similar to a regular B-form DNA. Exceptions are N4a and N5a where nonzero probabilities to a small extent are observed for regions corresponding to O4′-endo, and C3′-endo type conformations. The calculated probability distributions indicate that only three of the ten nucleotides (N1a, N2b, and N3b) substantially sample C3′-endo conformations corresponding to an A-form DNA (Figure 5). In addition to these three nucleotides, N4a and N5b exhibit a marginal propensity to convert to C3′-endo conformation. The temperature dependence on the structural transition observed in Figure 4 is contributed mainly by N2b and N3a. At 300 K, maximum probabilities are observed for the sampling of O4′-endo conformations. However at 360 K, sugar moieties of N2b and N3a are mostly found in the C3′-endo and C2′-endo conformations respectively. It is to be noted that the qualitative difference in the distributions of the pseudorotation angle corresponding to N3a and N3b of AZQ between the two different simulations at 360 K are consequence of base opening events observed in 360-2.



Figure 7. (a) Protein-DNA interactions involving the hydrophobic residues. The four hydrophobic residues are given in red, interacting nucleotides in gray, and the rest of the protein as cartoon representation in green. (b) Probability distributions corresponding to the protein-DNA interaction distances shown above (see text for more details).

TABLE 3: Mean Interaction Energies of the Central Six Base Pairs (kcal/mol) of the DNA Molecules Present in the Two Binary Complexes Obtained from Different MD Simulations DNA (Alone)

DNA (Complexed)

AZP

300 K

360 K

420 K

480 K

300 K

360 K-1

360 K-2

420 K

480 K

C2G7 G3C6 A4T5 T5A4 C6G3 G7C2

-23.0 ( 0.2 -22.6 ( 0.2 -11.0 ( 0.0 -11.0 ( 0.0 -22.0 ( 0.1 -23.5 ( 0.0

-9.6 ( 5.2 -16.2 ( 4.1 -10.6 ( 0.0 -10.6 ( 0.1 -17.9 ( 4.2 -18.6 ( 4.6

-4.1 ( 3.9 -6.0 ( 5.0 -2.3 ( 2.3 -1.8 ( 1.6 -3.8 ( 3.8 -1.7 ( 1.6

0.0 ( 0.0 0.0 ( 0.0 0.0 ( 0.0 0.0 ( 0.0 -0.3 ( 0.2 0.0 ( 0.0

-22.3 ( 0.1 -22.4 ( 0.2 -10.8 ( 0.1 -11.0 ( 0.0 -22.0 ( 0.1 -23.5 ( 0.0

-22.0 ( 0.2 -21.6 ( 0.1 -10.7 ( 0.0 -10.8 ( 0.0 -21.4 ( 0.2 -21.8 ( 0.3

-20.7 ( 2.9 -22.2 ( 0.1 -10.7 ( 0.1 -10.7 ( 0.0 -22.1 ( 0.2 -17.8 ( 2.4

-6.1 ( 5.1 -9.2 ( 4.4 -4.0 ( 2.3 -4.9 ( 2.6 -10.8 ( 5.6 -1.9 ( 1.6

0.0 ( 0.1 0.0 ( 0.0 -0.2 ( 0.2 -2.0 ( 0.6 -3.9 ( 1.5 -1.7 ( 0.7

AZQ T2A7 A3T6 A4T5 T5A4 T6A3 A7T2

300 K -10.9 ( 0.0 -10.9 ( 0.0 -10.6 ( 0.1 -10.7 ( 0.0 -11.0 ( 0.0 -11.2 ( 0.0

360 K 0.0 ( 0.0 -2.0 ( 1.2 -3.3 ( 2.4 -3.3 ( 2.4 -3.0 ( 2.4 -1.8 ( 1.6

420 K -0.2 ( 0.1 0.0 ( 0.0 0.0 ( 0.0 -0.1 ( 0.1 0.0 ( 0.0 0.0 ( 0.0

480 K 0.0 ( 0.0 -0.1 ( 0.1 0.0 ( 0.0 -0.2 ( 0.2 -0.1 ( 0.1 -0.2 ( 0.1

300 K -11.0 ( 0.0 -10.8 ( 0.0 -10.8 ( 0.1 -11.0 ( 0.0 -11.2 ( 0.0 -11.2 ( 0.0

360 K-1 -6.6 ( 0.6 -9.5 ( 0.5 -10.1 ( 0.2 -9.5 ( 0.4 -9.4 ( 0.8 -7.1 ( 0.8

360 K-2 -4.7 ( 1.8 -2.9 ( 1.6 -9.8 ( 0.4 -10.5 ( 0.1 -10.5 ( 0.1 -9.7 ( 0.7

420 K 0.0 ( 0.0 0.0 ( 0.0 0.0 ( 0.0 0.0 ( 0.0 0.0 ( 0.0 -0.2 ( 0.2

480 K 0.0 ( 0.0 0.0 ( 0.0 0.0 ( 0.0 0.0 ( 0.0 -0.3 ( 0.3 0.0 ( 0.0

The variability of the kink in the DNA structures was examined by calculating the “roll” helical parameter. Such an analysis was not done on the trajectories obtained at 420, and 480 K since the helical structure is lost. The probability distributions of the roll angle of the DNA in the protein-DNA complexes (300, 360-1/2), and for DNA alone (300 K) are given in Figure 6. The roll angle values calculated for the X-ray crystal structures are 59.9°, and 57.7° for AZP and AZQ, respectively. The distributions calculated for AZP are similar at the two temperatures no temperature dependence on the kink

angle, and agrees well with the angle obtained for the experimental structures. However, in AZQ, significant probability is observed at around 30° only at 360 K in both of the MD simulations. Visualization of the trajectories indicates that partial base pair opening of A3T6 and A4T5 allows for such a reversible relaxation in the kink. 3.3. Protein-DNA Interactions. Initially, the interaction energies between the protein, and the bases and backbone of each of the five nucleotides pairs shown in Figure 5 were calculated (Table S4 in Supporting Information). The reported

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interaction energies are not directly comparable to the experimental thermodynamic data, since energies obtained from experimental studies accounts for the desolvation energies of the DNA and protein, deformation energy corresponding to the DNA, etc.6,7 In contrast, the interaction energies calculated here are in the context of the protein-DNA complex. For any given nucleotide, the interaction energies of the backbone are 1-2 orders of magnitude more favorable than that of the base part. Comparison of the electrostatic and van der Waals contribution to the overall energies indicate that the interaction energies are dominated by electrostatic energies arising out of the interaction between the phosphate group of the DNA backbone and the positive residues of the protein (LYS/ARG). Thus, the DNA sequence nonspecific binding ability of Sac7d is a consequence of the extensive DNA backbone-protein contacts. McAfee et al. have shown that Sac7d binds to GC rich sequences strongly compared to the AT rich sequences.20 The interaction energy calculations show that such differences in the extent of binding are mostly likely due to the differential structure, intrinsic stability, and dynamics of the DNA due to the sequence differences, and not due to the direct participation of the bases themselves in binding. Other methods such as free energy decomposition analysis based on the Generalized Born model may also be used to further analyze the protein-DNA interactions, and the solvation effects in detail.47-50 The importance of the role of certain residues of Sac7d has been investigated by spectroscopic techniques. On the basis of a database survey of DNA minor groove binding proteins, Zhurkin and co-workers have shown that hydrophobic residues are crucial in B- to A- transitions in DNA in the bound form.51 In this section, we examine such contribution of hydrophobic interactions that are involved in DNA binding. Bedell et al. have shown that the surface tryptophan residue is crucial for DNA binding and for protein stability.19 W24A mutation of Sac7d leads to about 300-fold decrease in the DNA binding affinity. Examination of the crystal structure of Sac7d-DNA complexes reveals a single hydrogen bond with the DNA, and involves in hydrophobic packing of the sugar moiety of N3a (Figure 7a). At 300 K, the hydrogen bonding involving W24 is stable in most of the MD simulation time (Figure 7b). However, in all the four MD simulations at 360 K, the corresponding distance samples a longer value. In contrast, the probability distribution corresponding to the hydrophobic interaction between N3a and the side chain of W24 exhibits a very stable contact in all the simulations at both the temperatures. Therefore, W24 is proposed to be crucial for packing against the DNA backbone, and the hydrogen bond formed by W24 may not be essential for high DNA binding affinity. Notably, Bedell et al. have also shown that W24F exhibits only 20-fold lower binding affinity compared to the 300-fold lower binding affinity of W24A with respect to the wild type.19 In addition to W24, three other hydrophobic residues (V26, M29, and A44) are involved in hydrophobic contacts with the DNA (Figure 7a). V26 and M29 have shown to be important for the intercalation that causes a sharp kink in the DNA.7 The probability distributions of the minimum distances between the nonhydrogen atoms of the DNA bases (N1a, N2a, N1b, and N2b) and these two residues (V26 and M29) exhibit strong contacts. Similarly, A44 makes a strong contact with the sugar ring of N2b (Figure 7b). This interaction is responsible for the strong preference for C3′-endo, and O4′endo, and no preference for C2′-endo conformations by the sugar moiety, which probably contributes to DNA kinking.

Priyakumar et al. 4. Conclusions MD simulations were employed to investigate the ability of Sac7d protein to enhance the thermal stability of DNA via protein-DNA interactions, and to study the structural transitions involved. The protein structures showed little or no variation of their structure, and dynamics at 300 and 360 K in all six simulations. In contrast, DNAs are more flexible near the organism’s growth temperature, and were found to exhibit enhanced base opening, especially those that are not in contact with the protein. While the DNA is stabilized at 300 and 360 K, it undergoes denaturation at higher temperatures (420 and 480 K) even in presence of the protein. Within the binary complex, the DNA molecules were found to undergo B- to A-form transitions, which were facilitated by the hydrophobic residues in the binding site. Detailed analysis of the sugar puckering reveals that only 4-5 nucleotides sample C3′-endo regions, and sampling of such conformations was found to increase with temperature. Multiple MD simulations support the proposed model that Sac7d stabilizes up to five base pairs including the ones that are part of the kinking site. Nonspecific DNA binding ability of Sac7d is proposed to be mainly due to the extensive interaction with the backbone of the DNA, and the resultant strong electrostatic interactions. The differential binding of GC vs AT rich sequences previously reported are proposed to be due to the inherent structural and dynamic differences of the DNA molecules rather than the ability of the DNA bases to take part in binding. Acknowledgment. Department of Atomic Energy (Govt. of India), Department of Science and Technology (Govt. of India), and International Institute of Information Technology, Hyderabad are acknowledged for financial assistance. Supporting Information Available: Tables of detailed rmsd calculations on the DNA, percentage of base open states, inter and intrastacking interactions energies, and protein-DNA interactions, and figures of rmsd, contact maps, and base pair distances. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) White, M.; Bell, S. Trends Genet. 2002, 18, 621–626. (2) Sandman, K.; Reeve, J. N. Curr. Opin. Microbiol. 2005, 8, 656– 661. (3) Vieille, C.; Zeikus, G. Microbiol. Mol. Biol. ReV. 2001, 65, 1–43. (4) Robinson, H.; Gao, Y.; McCrary, B.; Edmondson, S.; Shriver, J.; Wang, A. Nature 1998, 392, 202–205. (5) Mukherjee, A.; Sokunbi, A. O.; Grove, A. Nucleic Acids Res. 2008, 36, 3956–3968. (6) Peters, W.; Edmondson, S.; Shriver, J. J. Mol. Biol. 2004, 343, 339–360. (7) Peters, W.; Edmondson, S.; Shriver, J. Biochemistry 2005, 44, 4794– 4804. (8) Demirjian, D.; Mor s-Varas, F.; Cassidy, C. Curr. Opin. Chem. Biol. 2001, 5, 144–151. (9) Haki, G.; Rakshit, S. Bioresour. Technol. 2003, 89, 17–34. (10) Niehaus, F.; Bertoldo, C.; Ko¨hler, M.; Antranikian, G. Appl. Microbiol. Biotechnol. 1999, 51, 711–729. (11) Unsworth, L.; van der Oost, J.; Koutsopoulos, S. FEBS J. 2007, 274, 4044–4056. (12) Chen, C.-Y.; Ko, T.-P.; Lin, T.-W.; Chou, C.-C.; Chen, C.-J.; Wang, A. H.-J. Nucleic Acids Res. 2005, 33, 430–438. (13) Ko, T.-P.; Chu, H.-M.; Chen, C.-Y.; Chou, C.-C.; Wang, A. H.-J. Acta Crystallogr., Sect. D 2004, 60, 1381–1387. (14) Su, S.; Gao, Y.-G.; Robinson, H.; Liaw, Y.-C.; Edmondson, S. P.; Shriver, J. W.; Wang, A. H. J. J. Mol. Biol. 2000, 303, 395–403. (15) Gao, Y.; Su, S.; Robinson, H.; Padmanabhan, S.; Lim, L.; McCrary, B.; Edmondson, S.; Shriver, J.; Wang, A. Nat. Struct. Mol. Biol. 1998, 5, 782–786.

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Molecular Simulations on the Thermal Stabilization of DNA by

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