Atomistic Simulation of Stacked Nucleosome Core Particles: Tail

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Atomistic Simulation of Stacked Nucleosome Core Particles: Tail Bridging, the H4 Tail and Effect of Hydrophobic Forces Suman Saurabh, Matthew A Glaser, Yves Lansac, and Prabal K. Maiti J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b11863 • Publication Date (Web): 02 Mar 2016 Downloaded from http://pubs.acs.org on March 7, 2016

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The Journal of Physical Chemistry

Atomistic Simulation of Stacked Nucleosome Core Particles: Tail Bridging, the H4 Tail and Effect of Hydrophobic Forces Suman Saurabh1, Matthew A. Glaser2, Yves Lansac3, 4,* and Prabal K. Maiti1,* 1

Center for Condensed Matter Theory, Department of Physics, Indian Institute of Science, Bangalore, India

2

Department of Physics and Liquid Crystal Materials Research Center, University of Colorado, Boulder, CO 80309, USA 3

GREMAN, Université François Rabelais, CNRS UMR 7347, 37200 Tours, France

4

School of Materials Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 61005, Korea

*To whom correspondence should be addressed. Email: [email protected] or yves.lansac@univ-

tours.fr

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ABSTRACT We report the first atomistic simulation of two stacked Nucleosome Core Particles (NCPs), with an aim to understand, in molecular detail, how they interact, the effect of salt concentration and how different histone tails contribute to their interaction, with a special emphasis on the H4 tail, known to have the largest stabilizing effect on NCP-NCP interaction. We do not observe specific K16mediated interaction between the H4 tail and the H2A-H2B acidic patch, in contrast to the findings from crystallographic studies, but find that the stacking was stable even in the absence of this interaction. We perform simulations with the H4 tail (partially/completely) removed and find that the region between LYS-16 and LYS-20 of the H4 tail holds special importance in mediating interNCP interaction. Performing similar tail-clipped simulations with the H3 tail removed, we compare the roles of the H3 and H4 tails in maintaining the stacking. We discuss the relevance of our simulation results to the bilayer and other liquid crystalline phases exhibited by NCPs in vitro and, through an analysis of the histone-histone interface, identify the interactions that could possibly stabilize the inter-NCP interaction in these columnar mesophases. Through the mechanical disruption of the stacked nucleosome system using steered molecular dynamics (SMD), we quantify the strength of inter-NCP stacking in the presence and absence of salt. We disrupt the stacking at some specific sites of inter-nucleosomal tail-DNA contact and perform a comparative quantification of the binding strengths of various tails in stabilizing the stacking. We also examine how hydrophobic interactions may contribute to the overall stability of the stacking, and find a marked difference in the role of hydrophobic forces as compared to electrostatic forces in determining the stability of the stacked nucleosome system.


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INTRODUCTION Two meter long eukaryotic DNA needs to be compacted tightly to be accommodated inside the cellular nucleus, which is of the order of 10 µm in diameter.1-3 At the first level of this compaction, 147 bp long DNA wraps around a protein octamer consisting of two copies each of four different kinds of histone proteins (H3, H4, H2A and H2B), resulting in a structurally dynamic1 DNA-protein complex known as the Nucleosome Core Particle (NCP). These NCPs, connected by linker DNA and compacted further, form the chromatin fiber. As DNA is negatively charged and the histone octamer has a net positive charge, the interactions involved in the formation of this complex are predominantly electrostatic. The strong electrostatic interaction compensates for the energy gain and entropy loss due to DNA bending and facilitates an overall gain in free energy. Bending of nucleosomal DNA is aided by the insertion of histone arginines into the DNA minor groove.4-6 The nucleosomal DNA can be categorized as mildly bent, as the deviation of its helical parameters from those of free B-DNA is very small in comparison to many other DNA-protein complexes.7 The histone proteins have a structured folded region and an unstructured region known as tail which protrudes out of the NCP. The histone tails belong to a class of proteins known as Intrinsically Disordered Proteins (IDPs).8-9 Such proteins do not fold to a unique three dimensional structure on their own, but do so when they bind to other macromolecules, gaining the ability to perform structure-based functions in addition to accommodating various post-translational modifications important for cellular processes.10-11 NCPs have been extensively studied in the recent years through experiments,4,

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atomistic26-29

(reviewed in 27) and coarse-grained30-35 simulations as well as theory.36-42 The role of histone tails in maintaining a stable nucleosome has been investigated.26 Histone tails are involved in inter- and intranucleosomal contacts22, 43-45 and contribute in an additive manner to the oligomerization of nucleosomal arrays21. H3 and H4 tails play a major role in moderately folding nucleosomal arrays,21 whereas strongly folded structures are achieved with a contribution from all the histone tails.46 Removal of H2A and H2B tails is known to have an impact on NCP reconstitution.13 Experimental studies reveal that the H4 tail holds comparatively higher importance in inter-nucleosome interaction as compared to other histone tails. Dorigo et. al.,16 performed self-assembly experiments along with analytical ultracentrifugation and showed that the removal of H4 tail precludes folding of nucleosomal arrays, while deletion of any other tail does not affect the process. Covalent modifications in the histone core and the tails, known as post-translational modifications, are known to aid in chromatin remodeling.47-55 The effects of post-translational 3 ACS Paragon Plus Environment


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modifications, such as lysine acetylation,48, 50-53, 55, 56 on the conformational behavior of the tails and the stability of chromatin have been thoroughly studied. Papoian and Potoyan,50 using atomistic MD simulation, showed that acetylation of LYS-16 residue of the H4 tail leads to an enhanced coiling of the tail in the vicinity of a DNA even though acetylation reduces its positive charge. The effect originates from the fact that acetylation turns the charged and polar lysine side chain into an uncharged and hydrophobic one leading to the collapse of the H4 tail into a coiled structure. Their work points towards the important role of hydrophobic forces, in addition to electrostatic forces, in determining tail conformation and hence in maintaining the chromatin structure. Yang et. al.,55 using replica exchange MD showed how the alpha-helical propensity of the region between residues 15 to 21 of the H4 tail gets reduced as a result of LYS-16 acetylation. Early studies on NCPs were mostly experimental in nature. After the determination of the X-ray crystal structure of the NCP4,

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and the growth in computational power, MD simulations started playing an

important role in providing complementary insights at a microscopic level. Recently, using molecular replacement techniques, the crystal structure of a tetranucleosome has also been determined.24 Such structures aid in better understanding of the chromatin structure. So far, all the atomistic simulations have focused on the behavior of a single NCP. Here, we simulate two NCPs stacked on top of each other to investigate how tails act as mediators in inter-nucleosome interaction and to what extent different tails contribute in holding the NCPs together, with a special emphasis on the H4 tail. Korolev and Nordenskiold57 performed MD simulation of a system of DNA fragments and H4 tails and showed interactions between the tails and DNA with the H4 tails mediating DNA-DNA interaction. Our simulations involving two NCPs clearly show such interactions of the positively charged residues of various tails of an NCP with DNA and histone core of the native and close-by NCP. We further dissect the roles of different regions of the H4 tail sequence in tail-bridging by clipping the H4 tail at various positions along its sequence. The nucleosome crystal structures show the H4 tail in a bridging conformation,4 interacting with the H2A-H2B acidic patch of a close-by nucleosome. The binding of the LYS-16 residue of the H4 tail at this negatively charged region is considered as the precursor for chromatin condensation. H2A variants with different degrees of acidity of the acidic patch, resulting from a mutation at any site belonging to the patch, lead to differently folded chromatin fibers.3, 58 The acidic patch is also the binding site for many chromatin remodeling proteins like HMGN2 and HP1 which compete with each for binding.59 In some regions of the chromatin, other proteins may win over the H4 tails in a bid to occupy the acidic-patch binding site. In case of an unavailability of the acidic patch for binding, it is most likely that the H4 tail binds along the nucleosomal DNA. The initial relative orientation of the two nucleosomes 4 ACS Paragon Plus Environment

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in our simulations (with their dyad axes parallel) is such that the H4 tail of one NCP cannot properly approach the acidic patch on the other and rather binds its DNA. While we see some of the H4 tail residues interacting non-specifically with the acidic patch of the neighboring NCP, our simulations demonstrate that even in the absence of the specific K16-mediated H4 tail-acidic patch binding, two stacked nucleosomes can hold on strongly to each other. Among many possible NCP orientations, we have focused only on the head-to-head orientation of the nucleosomes (parallel dyads) because of its biological relevance. Consecutive NCPs in the 30-nm chromatin fiber are expected to exhibit a head-tohead orientation with a slight tilt.60 The recently developed crystal structure for tetranucleosome24 also depicts stacked NCPs in a head-to-head orientation. The head-to-head orientation is also the favored one for the in-vitro NCP bilayer phase.15, 18-20 We believe that the study of the effect of salt concentration and tail-truncation on the stacked NCPs oriented parallel to each other will provide important insights into the process of chromatin formation and stability. We also demonstrate the important role played by hydrophobic forces in determining the stability of the stacked NCPs. Our findings would help in answering questions related to the experimentally observed liquid crystal phases in nucleosomal systems,18-20, 42, 61-63 and other deeper questions related to the problem of chromatin compaction and its stability. MATERIALS AND METHODS Equilibrium molecular dynamics simulations Preparation of intact NCP system. The 1.9 Å resolution NCP crystal structure corresponding to PDB ID: 1KX5, determined by Luger et. al.,25 was used as the starting structure for the simulation, from which all Mg2+ ions and crystal waters were removed. All simulations were performed using AMBER1264 and the systems were prepared using its xleap module. Amber force field ff10, which incorporates the parmbsc0 correction65 for DNA along with ff99SB66 parameters for proteins, was used to describe the nucleosome. TIP3P model was used for water while the monovalent ions were described using the Cheatham parameter set.67 To begin with, two nucleosomes were kept stacked on top of each other at a center of mass distance of 60 Å, with their dyad axes parallel to each other (Figure 1). The system was solvated with TIP3P water layer of 15 Å in all three directions. A total of 288 Na+ ions were then added to neutralize the system. The system contained 470226 atoms (AT-0). Here, AT stands for “All Tails”, indicating that all the histone tails are intact, while 0 refers to the added salt concentration. Another variant of this system was prepared with a NaCl concentration of 150 mM by adding 488 Na+ and an equal number of Cl- ions (AT-150). This system contained 469201 atoms. The details of these simulated systems are given in Table 1.



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Preparation of systems with histone tails (partially/completely) removed. For simulating NCPs with no tails, all the histone tails were removed from the crystal structure. The nucleosomes were then kept at a center of mass distance of 60 Å. Water was added with a solvation layer of 15 Å in the x- and ydirections and 35 Å in the z direction. 456 Na+ ions were added for charge neutralization. The system contained 363764 atoms. The system is named NT-0, which stands for “No Tails” and no added salt (see Table 1). Two simulations were also performed with the H4 tail clipped at two different positions (see Tables 1 and S1). In one of the simulations the H4 tail was removed from SER-1 to LYS-20 (H4-20-150) (here H4 is the tail that has been clipped, 20 is the clipping position and 150 refers to monovalent salt concentration. Similar naming scheme has been used for naming other tail-clipped simulations) and in the second one from SER-1 to ALA-15 (H4-15-150). For H4-20-150, 316 Na+ ions were added for charge neutralization and 488 Na+ and Cl- ions were added to achieve simulation condition corresponding to a salt concentration of 150 mM. Similarly, for H4-15-150, 308 Na+ ions were added for charge neutralization and 488 Na+ and Cl- ions were added for attaining a salt concentration of 150 mM. A variant of H4-20-150 was simulated at zero salt (H4-20-0). Two more simulations were performed at zero salt (H3-40-0) and 150mM salt (H3-40-150) with the H3 tail removed at the same position as that for the “No Tails” simulation (see Table 1). The system required 340 Na+ ions for charge neutralization. 326 Na+ and an equal number of Cl- ions were added to attain a salt concentration of 150mM for this system. Simulation Protocol. The systems described above were minimized for 1000 steps using steepest descent minimization method followed by 2000 steps of conjugate gradient minimization. During minimization, all the solute atoms were held fixed to their starting co-ordinates using harmonic constraints with a force constant of 500kcal/mol/Å2. This allowed water molecules and ions to re-organize and eliminate unfavorable contacts with protein and DNA. After this, 5000 steps of conjugate gradient minimization were performed, using a force constant that decreases from 20 kcal/mol/Å2 to 0, with a reduction of 5 kcal/mol/Å2 every 1000 steps. After minimization, the systems were heated from 0K to 300K within 40 ps while the solute was held fixed using harmonic constraints with a force-constant of 20kcal/mol/Å2. SHAKE68 constraints were applied to all the bonds involving hydrogen with a tolerance of 5×10-4Å. Temperature regulation was achieved using Berendsen thermostat69. A temperature coupling constant of 0.5 ps was used. After heating, a 500 ps long NPT simulation was performed by employing Berendsen barostat69 with a pressure relaxation time of 0.5 ps. Finally, 100 ns long NVT simulation was performed using a temperature coupling constant of 1ps. The resulting MD trajectories were visualized using VMD.70 The system constituted by the two solvated stacked NCPs is shown in Figure 1(A). In the discussion that follows, one of the NCPs (colored red) is called NCP1 while the other one (colored yellow) is named 6 ACS Paragon Plus Environment

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NCP2 (figure 1(B)).The N-terminal tails of the NCPs are named according to the convention NCP . For example the tail belonging to the first of the two H3 histones of NCP1 will be ( HISTONE ) TAILNo .

called (H3)11 . The other H3 tail of NCP1 will thus be called (H3)12 . The DNA belonging to NCP1 will be called DNA1 and the other one DNA2. A similar naming scheme will be used while referring to protein core as a whole (HISTONE1 and HISTONE2). A cartoon of the 2-NCP system with the different tails labeled is shown in Figure 1(B). NCP1 and NCP2 can be differentiated from each other if one notices that those H4 tails of the two NCPs that lie in the region between the two, and hence can take part in tailbridging, are different. (H4)11 of NCP1 faces NCP2 whereas (H4)22 of NCP2 faces NCP1. One can notice that H3 and H2B tails emerge out of the NCPs between the gyres of the DNA super-helix while H2A and H4 tails do so at the flat surface of the NCP. The properties of various histone tails are listed in Table S1 of Supporting Information (SI). Steered molecular dynamics simulations Constant velocity pulling simulations for moving the two NCPs apart were performed using NAMD71 using the same force field parameters as used for the above mentioned equilibrium simulations. The initial structures for the pulling runs were generated by performing a 500 ps long NPT simulation, using NAMD, on the final structures obtained from the AMBER runs. A similar protocol for initial system preparation was used for the site-specific pulling simulations (see Results and Discussion). The pulling runs were performed at different velocities ranging from 0.003 Å/ps to 0.03 Å/ps. The force constant used for each SMD atom was 5 kcal/mol/Å2. NCP1 was held fixed by keeping three atoms belonging to DNA1 fixed in space. NCP2 was pulled at the center of mass of 4 chosen atoms (SMD atoms). The SMD atoms belong to DNA2 and each one of them is located close to a tail (NCP1)-DNA2 binding site (See Results and Discussion). In the site-specific pulling simulations, NCP1 was again held fixed as described above. In addition to that, NCP2 was held fixed at a particular site and pulled away from NCP1 at another specific site, so that some particular contacts are broken while others stay intact (see Results and Discussion for details). Calculation of atomic contacts We have calculated the residue-wise inter-nucleosomal tail-mediated atomic contacts. An internucleosomal tail-mediated contact is said to exist if any atom of a residue belonging to a tail falls within 3 Å of an atom belonging to the neighboring NCP. The number of contacts is equal to the number of such atomic pairs. The tail-DNA and tail-protein contacts are calculated separately. The total number of tailmediated inter-NCP contacts can be calculated as a sum of the two. 7 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION The NCPs remained stable throughout the 100 ns long simulations. To quantify the stability and the amount of structural deviation from the starting structure, we have calculated the RMSD for the NCP and also for protein and DNA separately, with respect to the initial minimized structure, and have plotted them in figure 2. We find that the structural deviation of DNA is low with a RMSD of 3 Å. Protein RMSD is rather large (~ 9 Å) and a large part of it is contributed by the histone tails, which undergo large conformational changes during the simulation, starting from a relatively straight conformation to a collapsed one. The RMSD of the NCPs is around 7 Å. The RMSD in the presence and absence of salt attain very similar values (see Figure2). Inter-NCP stacking and the effect of salt The time evolution of the distance between the two NCPs is shown in Figure 3(A). We observe that the inter-NCP distance (~56 Å) in presence of salt (AT-150) is lower than that in the absence of salt (AT-0), an effect of salt screening the electrostatic repulsion between the two NCPs. Nevertheless, the distance in absence of salt seemed to saturate at a value of around 62 Å. It must be noted that NCPs kept far apart do not come together in absence of salt or even in presence of monovalent ions.15,

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Our initial

conformation, where the NCPs were placed very close to each other, and the fact that the conformational changes in tails occur at time-scales much smaller than the time-scales of significant translational displacement of NCPs, ascertain that the NCPs stay in close proximity even in the absence of salt. Before the NCPs can separate considerably due to electrostatic repulsion, the tails of one NCP form contacts with the DNA and protein of the other, and arrest the increase in inter-NCP distance. When all the tails of both the NCPs were removed in the absence of salt (NT-0), it was observed that the two NCPs fly apart along the long diagonal of the simulation box as shown in Figure 3(B). This result suggests that the histone tails hold the NCPs together in agreement with the various experimental findings44. Role of various tails in Tail-Bridging H4 Tail The H4 tail has a very high positive charge density. It is the smallest tail in terms of sequence length, but holds the highest importance in stabilizing the chromatin fiber.16 In our simulations at 150 mM NaCl (AT150), we observe that the H4 tail forms the largest number of inter-NCP contacts. The residues of the H4 tail, especially those in the stretch between LYS-16 to ARG-23, participate in a wide range of interactions with DNA and histone core of the neighboring NCP (see Figure 4). Positively charged residues interact electrostatically with the DNA backbone and also form strong salt-bridges with protein residues of the 8 ACS Paragon Plus Environment

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histone core, in addition to hydrogen bonds with the DNA bases and backbone. ARG-17 of (H4)11 is partially inserted into DNA2 minor-groove giving rise to strong stabilizing interactions (see Figure 4). The molecular details of the interaction are shown in Figure S1 of SI. HIE-18 of (H4)11 also forms contacts with DNA2 backbone. In Figure 5(A), we show the number of inter-NCP atomic contacts formed by the residues belonging to (H4)11 and (H4)22 with NCP2 and NCP1 respectively. The data is averaged over 200 configurations corresponding to the last 4 ns of the MD trajectory at 150mM salt concentration. One observes that (H4)11 forms large number of contacts with DNA2. (H4)22 has more contacts with the protein of NCP1 which include residues from (H2A)11 (see Figure S2 of SI). The behavior of (H4)22 is complicated because of the presence of (H2A)11 facing it, so, we expect the major stabilizing role to be played by (H4)11. There are some contacts between the basic residues of the H4 tail of one NCP and the acidic residues of the histone core of the other NCP. LYS-20 of (H4)11 forms salt-bridge with ASP-72 belonging to core histone H2A of NCP2 (marked as Saltbridge-1) (see Figure 4 and Table S2 of SI). LYS-8 and ARG-23 belonging to (H4)22 form salt bridges with GLU-56 and GLU-64 of histone H2A (NCP1) respectively, both belonging to the H2A acidic patch (see Figure 4). These two salt bridges are marked as Saltbridge-2 and Saltbridge-4 respectively. LYS-8 forms one more salt bridge with GLU-110 of histone H2B of NCP1 which is marked as Saltbridge-3 (see Figure 4 and Table S2 of SI). Apart from these, all other contacts made by (H4)22 with HISTONE1 involve the residues belonging to (H2A)11 (see Figure 4 and Figure S2 of SI). The prominent salt-bridges at 150 mM salt concentration are listed in Table S2 of SI and the snapshots depicting the salt bridges are shown with it. Because of the way the nucleosomes are placed relative to each other, the acid-base interactions are scattered (see Figure 4), unlike the specific and localized LYS16-mediated interaction between the H4 tail and the H2A-H2B acidic patch, as observed by Luger et.al4. A similar observation has been made in coarse-grained simulations31 and also in experiments showing bilayer phase of NCPs18,

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where initially randomly

oriented NCPs self-aggregate with their dyad axes aligned parallel to each other. At low salt concentrations and small osmotic stress, the NCPs first aggregate into columns which in turn form a bilayer (lamello-columnar) phase with an increase in osmotic pressure. This polar ordering has been attributed to tail-bridging interactions between NCPs belonging to neighbor columns. At higher salt concentrations, the bilayer phase disappears and a hexagonal columnar phase is observed.40,

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In this

hexagonal columnar phase, the NCPs in a column are randomly oriented with no orientational correlation. In the bilayer phase a lysine mediated interaction between the H4 tail and the acidic patch is not feasible due to the head-to-head orientation of the NCPs (parallel dyad axes). In the hexagonal columnar phase,



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the random orientation of NCPs suggests an absence of such specific interactions, since such an interaction would definitely correlate the orientation of the NCPs in a column. Isolated NCP columns are also formed at high enough NCP concentrations even in the absence of condensing agents and the NCPs in such columns are also stacked with random orientations18. The crystal structure of the tetranucleosome24 also has NCPs stacked with dyad axes almost parallel leading to preclusion of the H4 tail-acidic patch interaction. Sinha et. al.72 in experiments involving nucleosomal arrays demonstrated that an involvement of specific H4 tail residues in an intra-array interaction with the acidic patch would affect the association of the arrays. The LYS-mediated interaction might be specific to the inter-NCP orientation as seen in the crystal structure 1KX5. Although, this interaction can have a strong stabilizing effect, our simulation results suggest that it is not necessary to ensure the stability of NCP stacking. Figure 5(B) shows the average number of contacts for (H4)11 in the absence of salt (AT-0). We observe that in the absence of salt, the (H4)11–DNA2 contacts are localized to a small region of the tail (ALA-15 to HIE-18). This happens because in the absence of salt, tails are strongly bound to their native NCP. Also, in presence of salt, the screening of electrostatic repulsion between the two NCPs allows the tail to easily bind to DNA2. Again we observe ARG-17 of (H4)11 inserted into a DNA2 minor groove forming large number of contacts. We observe that in absence of salt there are no contacts between the residues of (H4)22 and (H2A)11 like those observed at 150 mM NaCl and shown in Figure S2 of SI. In the no salt case (AT-0), the electrostatic repulsion between the two tails pushes them apart. As (H4)22 and (H2A)11 tails do not form contacts with each other in the absence of salt, their residues form large number of contacts with DNA1 and DNA2 respectively as shown in Figure 5(B) and later in Figure 7(B). H4-Tail clipping. With an aim to find the region in the sequence of the H4 tail that makes it so important in tail- bridging, we performed 2-NCP simulations with different regions of the H4 tail removed (see Materials and Methods and Table 1). In the first simulation, amino acids from SER-1 to LYS-20 were removed (H4-20-150) and in the second simulation, the tail was clipped from SER-1 to ALA-15 (H4-15150). We also simulated a zero salt variant of H4-20-150 (H4-20-0). We find that, for H4-15-150, where the first 15 residues of the H4 tail are absent, the inter-NCP distance is not affected significantly in comparison to the intact-NCP simulation at 150 mM (AT-150). But in case of H4-20-150, the inter-NCP distance starts fluctuating and attains higher values as shown in Figure 5(C). This tells us that the most important contribution to the strong binding between H4 tail and DNA comes from the region of the tail between LYS-16 and LYS-20. As soon as this region of the tail is removed, the NCP stacking shows instability. H4-20-0 also shows rather large instability in NCP-NCP stacking as is evident from the large inter-NCP distance (Figure 5(D)), much similar to the effect seen in Figure 3(B) for the simulation with 10 ACS Paragon Plus Environment

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no tails (NT-0). A part of the increase in inter-NCP distance for the H4-clipped simulations is due to the in-plane sliding of the NCPs with respect to each other. In the absence of the densely charged region between LYS-16 and LYS-20, the repulsion between DNA1 and DNA2 causes the NCPs to shift relative to each other in the stacking plane (Figure S3 of SI). Although this sliding is observed in all the simulations, the effect was more pronounced in case of H4-20-150. Consecutive NCPs in a column forming a hexagonal LC phase also depict similar stacking conformation.20 The region from LYS-16 to LYS-20, in addition to forming a large number of inter-NCP contacts, plays a very important role in charge screening owing to its high charge density. The inter-NCP distance for H4-15-150 is shown in Figure S4(A) of SI. The contacts between the part of (H4)11 present in H4-15-150 and NCP2 are shown in Figure S4(B) of SI. We observe that HIE-18 of (H4)11 shows an increase in the number of contacts made with DNA2 in case of H4-15-150 as compared to the intact NCP system (AT-150). ARG-23 of (H4)11 also forms contacts with DNA2 in case of H4-15-150 whereas there are no such contacts in case of the intact NCP system. On further investigation we find that in case of AT-150, HIE-18 and ARG-23 of (H4)11 form strong intra-tail hydrophobic contacts with ALA-15. We infer that the hydrophobic residues in the amino acid sequence of a tail would restrain the motion of positively charged and polar residues through intra-tail hydrophobic contacts. HIE-18 and ARG-23 side chains will have increased sampling volume when ALA-15 is removed from the sequence as this will lead to loss of some hydrophobic contacts. The inter-NCP distance in case of H4-15-150 remains very close to the one obtained for the intact NCP system (AT-150). Although the region between LYS-16 and LYS-20 is present in case of H4-15-150, the inter-NCP stacking is expected to be at least slightly disrupted since many other positively charged residues (among the first 15 residues in the sequence) are absent. The minimal effect on the inter-NCP distance resulting from the removal of the first 15 residues of the tail means that the loss in charge and inter-NCP contacts because of tail-clipping has been partially compensated through extra contacts formed by HIE-18 and ARG-23. We compare the conformations of the (H4)11 before tail-clipping (AT-150) to that of the part of the H4 tail present in H4-15-150 system. We note that for H4-15-150 system, the H4 tail (LYS-16 onwards), which contains mostly positively charged residues, becomes very globular, with all the positively charged side-chains pointing towards DNA2, as shown in Figure S4(C) and (D) of the SI. This conformational change might result from the removal of hydrophobic residues like ALA-15 and LEU-10. Figure S4(E) shows the intra-tail contacts between ALA-15 and ARG-23 of (H4)11 for the last 4 ns of the AT-150 simulation. When ALA-15 is removed, ARG-23 points towards DNA2 and forms contacts with it. ARG-23 lies very close to NCP surface. The presence of contacts between such a residue of NCP1 and 11 ACS Paragon Plus Environment


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NCP2 would lead to significant arrest in distance between the two NCPs. The contacts formed by HIE-18 with ALA-15 are shown in Figure S4(F). A similar re-orientation of the side chain on tail-truncation is also observed in case of HIE-18 which forms larger number of contacts with DNA2 in case of H4-15-150 as compared to the intact NCP (AT-150) system (see Figure S4(B)). LEU-10 of (H4)22 also forms hydrophobic contacts with other residues of the tail and stops the tail from extending towards NCP1. The release of positively charged and polar residues from intra-tail hydrophobic contacts and their reorientation towards the DNA of neighboring NCP affect the inter-NCP distance in case of H4-15-150 (see Figure S4(A)) and restrain it to be very close to that of the AT-150 system in magnitude. Tail bridging process is therefore controlled by electrostatic interactions with significant contribution coming from hydrophobic interactions. Other effects of hydrophobic interaction are described in Figure S5 of SI where one observes how the radius of gyration Rg of (H2A)11 changes as (H4)22 is clipped. The residues of intact (H4)22 shield the hydrophobic residues of (H2A)11. As the (H4)22 tail is clipped, the hydrophobic residues of (H2A)11 form clusters, to avoid interaction with water. This leads to a smaller Rg for (H2A)11 in case of both H4-20-150 and H4-15-150 as compared to the intact NCP simulation (AT-150). We next move on to discuss the role of H3 tail in tail-bridging. H3 Tail H3 tail has the longest amino acid sequence among all the histone tails and has the largest positive charge. But in our simulations, it did not seem to play a significant role in tail-bridging. In all the simulations reported in this work, H3 tail remained collapsed close to its native NCP and did not extend towards the close-by NCP to form contacts. The globular state, which the tail likes to be in, could possibly be due to its glycine-rich sequence, which would lead to a large entropy loss if the tail extends. It is worth noting that in all the intact 2-NCP simulations, we did not find a single instance where the H3 tail of an NCP moved towards the DNA of the near-by NCP and formed contacts with it. H3-Tail clipping. Experiments show that the removal of H3 tail does not have as significant an effect on inter-NCP interaction as compared to the case when the H4 tail is removed17. We performed a 100 ns long simulation at zero salt (H3-40-0) and 150 mM NaCl (H3-40-150), removing the H3 tail completely (see Materials and Methods and Table 1) to understand their possible indirect role in inter-NCP interaction. We plot the inter-NCP distance for the two systems in Figure 6(A) and (B). We find that, for the H3-tail clipped systems, the distance between the two NCPs stayed fairly close to that for the intact NCP systems. This is in contrast to the case when the H4 tail was clipped where the inter NCP distance attains very large values. This observation tells that the removal of H4 tail has a much larger effect on NCP-NCP


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stacking as compared to the removal of H3 tail. This difference is due to the mode of contribution of the H3 and H4 tail to the stability of the NCP stacking. H4 tail of one NCP forms direct contacts with the DNA and protein of the other, whereas the H3 tail contributes to the stability by reducing the effective negative charge of its native NCP, by covering its DNA. Thus, when the H3 tail is removed from the NCP, the extra counterions (compared to the intact NCP case) added to the system for chargeneutralization can mimic the role of H3 tail by balancing some of the negative charge of the NCP. On the contrary, the presence of these extra counterions cannot substitute for the specific contacts formed by H4 tails with the DNA of the neighboring NCP. This leads to the difference in inter-NCP distance in both cases. In both the H3-tail clipped systems we observe that the H4 tails form inter-NCP contacts and arrest the increase in inter-NCP distance. There have been many experimental studies14, 17, 21, 24 demonstrating the importance of histone tails in the formation of nucleosomal arrays and their oligomerization. Bertin et. al.14 showed the importance of H3 and H4 tails in inter-NCP interaction. They proposed an interaction model for nucleosomes and found that in order to explain the experimental structure factors obtained using SAXS at high enough salt concentrations they had to add an attractive term to their theoretical model. Whereas in the absence of H3 and H4 tails, a repulsive term alone was enough to explain the profiles. Differences between the roles of H3 and H4 tails have been experimentally studied by clipping the tails one by one. Qiu et. al.17 studied inter-nucleosome interaction using SAXS with H3 and H4 tails deleted in turn. While deletion of the H3 tail does not change the nature of the interaction, deleting the H4 tail turns the interaction from attractive to repulsive. This suggests that the H4 tail has site-specific internucleosomal interactions, whereas the H3 tail facilitates attraction between nucleosomes by screening the electrostatic repulsion between them. The results from our tail-clipped simulations along with those obtained from equilibrium simulations of intact NCPs, together demonstrate the sharp differences between the bridging roles of the H4 and H3 tails and support the experimental findings. The equilibrium simulations show no inter-NCP contacts formed by the H3 tails whereas the H4 tails form significant number of contacts. And hence the clipping of H4 tail affects the inter-NCP stacking in a more severe manner than the clipping of the H3 tail. The effect is visualized through difference in the inter-NCP distance plots for H3 and H4 tail clipped simulations. Next, we study the role of H2A tail in the interNCP interaction. H2A Tail We observe that (H2A)11, at 150mM salt, forms contacts with DNA2 through three of its residues namely THR-10, ARG-11 and ALA-12 (see Figure 4). In contrast, at zero salt concentration a larger region of the tail forms contacts with DNA2. We see contacts between them (H2A)11 and HISTONE2 (mostly the



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residues of (H4)22 ) at 150 mM salt (Figure 7(A)), but such contacts are absent at zero salt (Figure 7(B)), as discussed above (in the section for H4 tail), allowing (H2A)11 to form more contacts with DNA2. Unlike the H4 tail, a localized region of the sequence of the H2A tail participates in tail bridging. ARG-11 of (H2A)11 gets inserted into the minor groove of DNA2 and holds it strongly, while THR-10 forms hydrogen bonds with the DNA2 backbone. We observe direct and water-bridged inter-NCP hydrogen bonds between ARG-11 and the DNA2 bases. The molecular details of the interactions at 150 mM salt are shown in Figure S6 of SI. One observes that unlike the H4 tail, only a localized region of (H2A)11 (THR-10 to ALA12) forms contacts with DNA2. To estimate the energy difference between bound and unbound states of the THR-ARG-ALA tripeptide and NCP, we have performed MMGBSA calculations on the complex between NCP and the tri-peptide using MMPBSA.py73 module of AMBER12. The binding energy of the complex comes out to be -12.8 ± 4.2 kcal/mol, signifying strong complexation (thermal energy is 0.6 kcal/mol at room temperature). The strong binding suggests that arginine-minor groove interactions can provide significant stability to the inter-NCP stacking. The details of the calculation and the various contributions to the total binding energy are provided in Table S4 of SI. As mentioned above, in our simulations we observe interaction between tails in addition to the tail-DNA and tail-histone core interactions. The snapshots showing this interaction are shown in Figure S4 of SI. H2B Tail In our simulations the N-terminus of H2B tail does not participate in tail bridging. But we observe that LYS-122 at the extremity of the C-terminus of histone H2B (NCP1) faces outwards and is placed close to the flat face of NCP2. This lysine residue mediates contacts between NCP1 and NCP2 (see Figure 4). It forms hydrogen bonds with DNA2 bases, backbone and other protein residues belonging to the histone core of NCP2, and interacts electrostatically with the DNA2 phosphates, thus contributing strongly towards the stability of the stacking. This residue forms nearly 8 contacts on an average with NCP2, calculated over the last 4 ns of the MD trajectory (AT-150). Molecular details of the interaction of this residue are provided in Figure S7 of SI. The residue is also seen to form inter-NCP contacts in case of AT-0 system, though the number of contacts formed (on an average 4) is less than the number of contacts formed at 150mM salt. The same lysine residue also forms inter-NCP contacts in the various tail-clipped simulations described here. The stacking of NCPs on top of each other to form long columns acts as the precursor to the formation of liquid crystal phases. Leforestier et. al.18 demonstrated the existence of bilayers of NCP columns above a critical NCP concentration and at moderate ionic conditions. They attributed the attraction between the


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lateral faces of NCPs belonging to different columns to tail-bridging by N-terminal H2B tail. The face-toface attraction between NCPs in a given column in such delicate NCP mesophases may originate from the arginine-minor groove interactions discussed previously (section on H4 and H2A tail) and possibly also from the C-terminal lysine of the H2B histone described above. The hydrogen bonds and salt-bridges at the interface of the histone cores of the two nucleosomes would also play a very important role in stabilizing the NCP-NCP stacking in such phases. When proteins fold, the hydrophobic amino acid residues get buried in the interior protein core and the hydrophilic ones stay on the surface. The oppositely charged residues on the histone core surface form salt bridges with surface residues of the other nucleosome (Table S2 of SI). The hydrophilic amino acid residues (both neutral and charged) on the protein surface form inter-NCP hydrogen bonds with each other74 (see Table S3 of SI). When two hydrophilic residues are not within hydrogen bonding distance then water molecules mediate hydrogen bonds between them, as shown in Figure S8. The non-specific nature of these interactions would allow for a random orientation of the NCPs in a given column, as observed in experiments.18, 61 Mechanical Disruption of NCP-NCP Stacking We have also performed pulling simulations to understand how the stacking of the two NCPs gets disrupted. We kept NCP1 fixed, by fixing in space three atoms along DNA1, and pulled NCP2 at the center of mass of four atoms belonging to DNA2, each of which lie near a contact between histone tails of NCP1 and DNA2 (see Figure 8(D)). We found that, in most of the pulling runs all the contacts between NCP1-NCP2 break except the strong contacts between (H4)11 and DNA2. Even when the (H4)11 - DNA2 contacts break, they do so at the end of the pulling, after all other contacts have ruptured. The effect persists for a large range of pulling velocities (0.003 Å/ps to 0.03 Å/ps). Figure 8(A), (B) and (C) show the sequence of rupture events for five different NCP1-NCP2 contacts for three different pulling runs performed at a pulling velocity of 0.003 Å/ps at 150 mM salt. Rupture is marked by a sudden increase in distance between the center of mass of the residue belonging to NCP1 and the center of mass of a group of atoms of NCP2 near the region where the residue binds. During the course of pulling, the applied force keeps on rising, till the ARG-11( (H2A)11)-DNA2 contact breaks off, and then goes to zero, breaking remaining contacts in the process. The contacts formed by ARG-17 of (H4)11 remain intact throughout or break down only in the later stages of pulling, and this happens both in presence and absence of salt. This observation suggests that (H4)11 -DNA2 contacts mediated by ARG-17 are the strongest. Figure 8(D) shows various snapshots from one such pulling run.



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Effect of Salt. To examine the effect of salt on the strength of stacking, we also performed pulling simulations at two different velocities (0.003 Å/ps and 0.008 Å/ps) at zero and 150 mM salt concentration. We observe that the rupture force at zero salt is always lower than that at 150 mM salt. Force-extension plots are shown in Figure 8(E) and (F). This suggests a stronger binding in presence of salt due to electrostatic screening. The difference in rupture force will also have contribution from the different binding conformations of histone tails and DNA at these two salt concentrations. Three pulling runs were performed at each velocity. For v = 0.008 Å/ps, the rupture force at zero salt concentration comes out to be 1278 ± 53 pN and that at 150 mM salt concentration is 1402 ± 74 pN. For a lower velocity of pulling at 0.003 Å/ps, the rupture forces in absence and presence of salt are 915 ± 58 pN and 1216 ± 88 pN respectively. Figures S9 and S10 of SI show force-extension plots for all the pulling runs in presence and absence of salt at pulling velocities of 0.008Å/ps and 0.003Å/ps respectively. Disruption of NCP-NCP stacking at specific sites. In our simulations, we found that (H4)11 and (H2A)11 form most of the inter-NCP contacts. To quantify the difference in strengths with which these two tails hold DNA2, we performed two different kinds of pulling simulations. In one of them, we held fixed the region of NCP2 near the (H2A)11–DNA2 binding site (see figure 4, label 6) in addition to keeping NCP1 fixed and pulled NCP2 at the (H4)11 –DNA2 binding site (see figure 4, label 1) while doing the exact opposite in the other simulation. Here, the binding site of a given tail refers to the site where it forms the strongest contact with DNA2 (i.e. ARG11-DNA2 binding site for (H2A)11 and ARG17-DNA2 binding site for (H4)11). The pulling simulations were performed at a velocity of 0.008 Å/ps. The pulling details and snapshots are shown in Figure 9(A) and (B). We see a large difference in the first force peaks in both cases. For H2A tail, the rupture force is around 496 ± 32 pN, whereas for the H4 tail it is around 827 ± 43 pN. This quantifies the strength of the H4 tail binding to DNA2 as compared to the H2A tail binding. Force-extension plots for all the site-specific pulling runs are shown in Figure S11 of SI. The strongest contacts that both the tails form with DNA2 correspond to the ARG-DNA-minor groove contacts. We note that, even though ARG-11 of (H2A)11 is inserted more prominently into DNA-minor groove as compared to ARG-17 of (H4)11 (see Figure S2(A) and S6(A) of SI), the peak in the force is larger for the H4 tail binding site pulling. This result suggests a possible cooperative behavior among the residues belonging to the amino acid sequence of a given histone tail. The high value of the rupture force in case of (H4)11 binding site pulling would result from a large number of positively charged (H4)11 residues, (apart from ARG-17), forming contacts with the DNA, whereas only three residues of (H2A)11, including ARG-11 being involved in tail-DNA contacts. The large number of consecutively placed positively 16 ACS Paragon Plus Environment

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charged residues of

(H4)11 would co-operatively interact with DNA2 and make it harder to break the

contacts between them. This highlights the importance of a region of high charge density like the one from LYS-16 and LYS-20 of in

(H4)11 , tail-bridging and suggests that in addition to the total charge

of a tail, the distribution of the charge along its sequence is also a determinant of its importance in tailbridging. The breaking of the ARG17-DNA2 contact at the very end, when all other contacts have ruptured, while pulling the two NCPs apart (Figure 8), also points towards a possible cooperative behavior. The force-extension peaks and corresponding rupture events for H4-binding site pulling at a lower velocity of 0.0033Å/ps is shown in Figure S12 of SI. One can observe the sharp jump in force when contacts formed by ARG-17 with DNA2 break. The snapshots in Figure S12 show how the consecutively placed positively charged residues of the H4 tail interact cooperatively with DNA. CONCLUSION To summarize, using various equilibrium and non-equilibrium simulation techniques, we provide a microscopic picture of the important role played by various histone tails in chromatin stability. From our simulation results, it is clear that the electrostatic interactions play a dominant role, but are modulated by hydrophobic forces. Insertion of arginine residues from histone tails into DNA minor groove is one of the modes of contact between two NCPs. Various inter-NCP salt-bridges and hydrogen bonds (direct and water-mediated) are observed. These are expected to play an important role in stabilizing NCP columns which self-assemble and give rise to bilayers and other liquid-crystalline phases in-vitro. The residues from LYS-16 to LYS-20 are very important in the strong tail-bridging exhibited by the H4 tail. Clipping the H4 tail at LYS-20 makes the inter-NCP distance fluctuate strongly signifying that the NCP-NCP stacking is destabilized. Hydrophobic residues present in the tail sequence restrict the conformational freedom of positively charged residues, and clipping them makes the charged residues free to move around and participate in bridging, as seen in the case of ARG-23 and HIE-18 of (H4)11. From the behavior of different histone tails in equilibrium simulations, and under mechanical rupture, we can infer that various properties of a tail would contribute to the way it participates in tail-bridging. The charge of a tail as a whole is important, but the behavior also seems to depend on how the charge is distributed along the sequence. A densely charged region in a tail would lead to very strong electrostatic interactions with DNA and also lead to cooperative binding, resulting in stronger interactions. The H4 tail contains such a region between LYS-16 to LYS-20 and so, even though its linear charge density is similar to the one of the H2A tail, it shows a stronger binding to DNA through a dominant contribution from that region. The relative position of the tails in the histone core also influences its ability to contribute to stacking interactions. We observe that, H4 and H2A tails, which form most of the inter-NCP contacts, protrude out of the NCP from its flat surface. As a result, they directly face the neighboring NCP and are important in 17 ACS Paragon Plus Environment


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the formation of NCP columns through stacking interaction. In contrast, the H3 and H2B tails protrude out laterally from the DNA gyres and hence are more suited to mediate lateral interactions. McBryant et. al.75 reported a set of experimental studies on nucleosomal arrays with various modified histone tails. They studied the effect of tail charge density, length and position on the amount of MgCl2 required for condensing the arrays. In an interesting experiment they found that removing H4 tail increases the salt required for array condensation more than when H2B tail is removed but replacing H2B tail by H4 tail does not decrease the salt requirement. This suggests that H4 tail is more effective when it is at its “original” position than when it is shifted to a position where it protrudes out of the NCP from between the DNA gyres like the H2B tail. The NCP-NCP stacking was stable even in the absence of the specific binding of the H4 tail to the H2A-H2B acidic patch which points towards the possibility of alternative bridging mechanisms for the H4 tail in situations which demand a moderate opening-up of the chromatin fiber. Tails, in addition to interacting with the DNA of the nearby NCP, also interact among themselves. The tail-tail interaction is driven by hydrophobic and electrostatic forces and influences the stability of chromatin. Supporting Information Available The Supporting Information includes details of the histone tail amino acid sequence, atomistic details of the inter-nucleosomal tail-DNA interactions, tail-tail interaction, hydrogen bonds and salt-bridges across the core-core interface, force-extension plots and atomistic details corresponding to various steered MD simulations. Acknowledgement: We thank DAE, India, for financial support. PKM thanks USIEF for a Fulbright-Nehru Senior Research Fellowship that enabled his stay at the University of Colorado, Boulder where part of this work was carried out. This work was also supported by the Soft Materials Research Center under NSF MRSEC Grants DMR-0820579 and DMR-1420736. This work also utilized the Janus supercomputer, which is supported by the National Science Foundation (award number CNS-0821794) and the University of Colorado Boulder. The Janus supercomputer is a joint effort of the University of Colorado Boulder, the University of Colorado Denver and the National Center for Atmospheric Research. Janus is operated by the University of Colorado Boulder. We also thank the support from the Korea-India SAVI Program (2013K2A1B9066145) of the Korean National Research Foundation. YL thanks the Korean CCS 2020

Program and the Brain Pool Program of KOFST (121S-1-3-0380, 131S-1-3-0504 and 151S-1-31232) for support.


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44. Hizume, K.; Nakai, T.; Araki, S.; Prieto, E.; Yoshikawa, K.; Takeyasu, K. Removal of histone tails from nucleosome dissects the physical mechanisms of salt-induced aggregation, linker histone H1induced compaction, and 30-nm fiber formation of the nucleosome array. Ultramicroscopy 2009, 109, 868-873. 45. Mangenot, S.; Raspaud, E.; Tribet, C.; Belloni, L.; Livolant, F. Interactions between isolated nucleosome core particles: A tail-bridging effect? Eur. Phys. J. E 2002, 7, 221-231. 46. Annunziato, A. T.; Hansen, J. C. Role of histone acetylation in the assembly and modulation of chromatin structures. Gene Expr. 2000, 9, 37-61. 47. Tessarz, P.; Kouzarides, T. Histone core modifications regulating nucleosome structure and dynamics. Nat. Rev. Mol. Cell Biol. 2014, 15, 703-708. 48. Shogren-Knaak, M.; Ishii, H.; Sun, J. M.; Pazin, M. J.; Davie, J. R.; Peterson, C. L. Histone H4K16 acetylation controls chromatin structure and protein interactions. Science 2006, 311, 844-847. 49. Strahl, B. D.; Allis, C. D. The language of covalent histone modifications. Nature 2000, 403, 4145. 50. Potoyan, D. A.; Papoian, G. A. Regulation of the H4 tail binding and folding landscapes via Lys16 acetylation. Proc. Natl. Acad. Sci. USA 2012, 109, 17857-17862. 51. Liu, Y.; Lu, C.; Yang, Y.; Fan, Y.; Yang, R.; Liu, C. F.; Korolev, N.; Nordenskiold, L. Influence of histone tails and H4 tail acetylations on nucleosome-nucleosome interactions. J. Mol. Biol. 2011, 414, 749-764. 52. Loidl, P. Histone acetylation: Facts and questions. Chromosoma 1994, 103, 441-449. 53. Dion, M. F.; Altschuler, S. J.; Wu, L. F.; Rando, O. J. Genomic characterization reveals a simple histone H4 acetylation code. Proc. Natl. Acad. Sci. USA 2005, 102, 5501-5506. 54. Bowman, G. D.; Poirier, M. G. Post-translational modifications of histones that influence nucleosome dynamics. Chem. Rev. 2015, 115, 2274-2295. 55. Yang, D.; Arya, G. Structure and binding of the H4 histone tail and the effects of lysine 16 acetylation. Phys. Chem. Chem. Phys. 2011, 13, 2911-2921. 56. Struhl, K. Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 1998, 12, 599-606. 57. Korolev, N.; Yu, H.; Lyubartsev, A. P.; Nordenskiöld, L. Molecular dynamics simulations demonstrate the regulation of DNA-DNA attraction by H4 histone tail acetylations and mutations. Biopolymers 2014, 101, 1051-1064. 58. Bonisch, C.; Hake, S. B. Histone H2A variants in nucleosomes and chromatin: More or less stable? Nucleic Acids Res. 2012, 40, 10719-10741. 59. Kalashnikova, A. A.; Porter-Goff, M. E.; Muthurajan, U. M.; Luger, K.; Hansen, J. C. The role of the nucleosome acidic patch in modulating higher order chromatin structure. J. R. Soc., Interface 2013, 10, 20121022. 60. Kepper, N.; Foethke, D.; Stehr, R.; Wedemann, G.; Rippe, K. Nucleosome geometry and internucleosomal interactions control the chromatin fiber conformation. Biophys. J. 2008, 95, 3692-3705. 61. Livolant, F.; Mangenot, S.; Leforestier, A.; Bertin, A.; Frutos, M. d.; Raspaud, E.; Durand, D. Are liquid crystalline properties of nucleosomes involved in chromosome structure and dynamics? Philos. Trans. R. Soc., A 2006, 364, 2615-2633. 62. Mangenot, S.; Leforestier, A.; Durand, D.; Livolant, F. Phase diagram of nucleosome core particles. J. Mol. Biol. 2003, 333, 907-916. 63. Bertin, A.; Mangenot, S.; Renouard, M.; Durand, D.; Livolant, F. Structure and phase diagram of nucleosome core particles aggregated by multivalent cations. Biophys. J. 2007, 93, 3652-3663. 64. Case, D.; Darden, T.; Cheatham III, T.; Simmerling, C.; Wang, J.; Duke, R.; Luo, R.; Walker, R.; Zhang, W.; Merz, K.; et. al. AMBER 12; University of California: San Francisco, 2012. 65. Pérez, A.; Marchán, I.; Svozil, D.; Sponer, J.; Cheatham, T. E.; Laughton, C. A.; Orozco, M. Refinement of the AMBER force field for nucleic acids: Improving the description of α/γ conformers. Biophys. J. 2007, 92, 3817-3829.



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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1: System Details. S.no. SYSTEM NAME

DESCRIPTION

SALT CONC.

TAIL DETAILS

CLIPPING

SYSTEM SIZE (Atoms)

TAIL(s)

CLIPPING POSITION

1

AT-0

Two in-tact NCPs.

0 mM

470226

-

-

2

AT-150

Two in-tact NCPs.

150 mM

469201

-

-

H3

ARG-40

H4

ASN-25

H2A

PRO-26

H2B

TYR-34

3 NT-0

4

5

6

7

8

Two NCPs with no tails.

0 mM

363764

H4-20-150

Two NCPs with H4 tail removed upto LYS 20. 150 mM

469591

H4

LYS-20

H4-15-150

Two NCPs with H4 tail removed upto ALA 15. 150 mM

469552

H4

ALA-15

H4-20-0

Two NCPs with H4 tail removed upto LYS 20. 0 mM

470595

H4

LYS-20

H3-40-0

Two NCPs with H3 tail removed. 0 mM

289690

H3

ARG-40

H3-40-150

Two NCPs with H3 tail removed. 150 mM

422535

H3

ARG-40



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A

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B STACK

NCP2

NCP1

60 Å

Figure 1 (A) Two nucleosomes placed side-by-side at a distance of 60 Å and solvated in water. (B) A schematic showing the two NCPs, represented as cylinders, stacked on each other. The red NCP is NCP1 and the yellow one is NCP2. The position of various tails can be seen. One can note that they are placed with their dyad axes (brown arrows) parallel to each other.


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A

B

Figure 2: RMSD of different components of (A) AT-150 system and (B) AT-0 system with respect to their minimized structures. The RMSDs in presence and absence of salt are almost same. Largest contribution to the RMSD of the NCPs comes from the histone tails that undergo a large conformational change relative to the initial structure. Conformations of DNA and the structured regions of the histone core do not change much throughout the simulation.



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B

A

Figure 3: Distance between centers of mass of two nucleosomes for (A) AT-0 and AT-150 systems. The inter-NCP distance is slightly lower in the presence of salt than in its absence. (B) NT-0 system. The snapshots shown along with the plot show how the NCPs move away from each other, illustrating the important role played by the histone tails to maintain the inter-NCP stacking.


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6

7 5

8 3 1

2 NCP2 NCP1 4

Figure 4: Inter-nucleosomal interaction sites. 1. Interaction between (H 4)11 (shown in black on NCP1) and DNA2. The red region on DNA2 marks their binding region. 2. N-Terminal region of (H4)11 depicted in green and corresponding binding region on DNA2 (red). 3. Salt bridge between LYS-20 of (H4)11 and NCP2 core (Saltbridge-1). 4. Sites of interaction between (H4)22 and residues in and around the H2A/H2B acidic patch of NCP1 (Saltbridge-2 ( ), -3 ( ) and -4 ( )). 5. (H2A)11- (H4)22 interaction regions (Tail-Tail interactions). 6. Interaction between (H2A)11 and DNA2. The red region on DNA2 marks the binding region. 7. Interaction between LYS-122 (shown in orange) of H2B (NCP1) and the corresponding binding region on NCP2 (shown in green). 8. Saltbridge between HISTONE1 and HISTONE2 core residues (Saltbridge-5). Note: Blue regions are the regions forming core-core hydrogen bonds between NCP1 and NCP2. Regions involved in water- mediated hydrogen bonds are not shown. mark the exit points of (H4)11,

(H2A)11 and (H4)22 respectively. Pairs forming Saltbridges-1 to -5 are listed in Table S2 of SI.



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A

B

D

C

Figure 5: Average number of contacts of different H4 tails with DNA and histone core of the neighboring NCP for (A) 150 mM salt (AT-150) and (B) zero salt (AT-0). Time evolution of inter-NCP distance for the system wih H4 tail clipped at LYS-20 for (C) 150 mM salt (H4-20-150) and (D) zero salt (H4-20-0). The inter-NCP distance shows large fluctuations when the H4 tail is removed upto LYS-20, demonstrating the important role played by the H4 tail in stabilizing the stacked conformation. The difference in inter-NCP distance in presence and absence of salt as seen in (C) and (D) demonstrate the role of salt. The embedded snapshots show the conformation of the stack for the tail-clipped simulations along the time evolution. 28 ACS Paragon Plus Environment

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B

A

Figure 6: Time evolution of inter-NCP distance for the system wih H3 tail clipped at ARG-40 for (A) 150 mM salt (H3-40-150) and (B) zero salt (H3-40-0). One notes that the fluctuations on the removal of the H3 tail are not that prominent as is the case of H4 tail removal shown in figure 5, demonstrating the superior role played by the H4 tail in stabilizing the stacking. The embedded snapshots show the conformation of the stack for the tail-clipped simulations along the time evolution.



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A

B

Figure 7: Average number of contacts of (H2A)11 with DNA and histone core of the neighboring NCP for (A) 150 mM salt (AT-150) and (B) zero salt (AT-0). One notes that all the bridging contacts formed by the H2A tail in absence of salt are with DNA2. However in the presence of salt the tail forms contacts with protein residues belonging to (H4)22 . Such contacts are absent at zero salt due to the strong electrostatic repulsion between the two densely charged tails (see table S1).


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A

D

(H4)22 (H2A)11

B

(H2B)11

SMD atom

(H4)11 C E

F

Figure 8:(A) (B) and (C) Rupture points for three pulling runs at 0.003 Å/ps. The force-distance plots are shown in the background. Pairs forming Saltbridge-1 and Saltbridge-3 are listed in table S2 of Supporting Information and the positions of all the 5 binding sites considered are shown in Figure 4. Rupture is defined as a sudden increase in distance between a residue and its binding site. (D) NCP2 (yellow) being pulled apart with NCP1 (red) held fixed by fixing in space three equally spaced atoms on DNA1. The red spheres on DNA2 are the SMD atoms. NCP2 was pulled at different velocities (v) at the center of mass of the four SMD atoms along the line joining the centers of mass of the two NCPs. The pulling direction is 31 ACS Paragon Plus Environment


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shown. (E) Force v/s distance plot for one of the pulling runs at a velocity of 0.003 Å/ps at different salt concentrations. (F) Force v/s distance plot for one of the pulling runs at a velocity of 0.008 Å/ps at different salt concentrations. One notes that the rupture force, defined as the peak force in the forceextension plot, is larger in presence of salt.

A

(H2A)11

B

ARG-11 v

NCP2 fixed here Figure 9. (A) Snapshots for one of the site-specific pulling runs. NCP1 (red DNA) is fixed and NCP2 (yellow DNA) is being pulled at the site where ARG11 of (H2A)11 binds with DNA2, while keeping it fixed near (H4)11-DNA2 binding site. The figure shows the position of NCP2 with respect to ARG-11 at different times during the pulling run. ARG-11 is shown in mauve in the otherwise grey (H2A)11. The pulling direction is shown by an arrow on the right. The point of application of force is depicted as a red dot on DNA2. The site where NCP2 is fixed is also shown. (B) Comparison of force v/s distance plots for one of the three pulling runs at the (H4)11–DNA2 binding site with that of (H2A)11–DNA2 binding site at a pulling velocity of 0.008 Å/ps. The plot shows that it requires a larger force to break the (H4)11 –DNA2 contacts indicating stronger binding.


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TOC Graphic


Atomistic Simulation of Stacked Nucleosome Core Particles: Tail

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