Structural dynamics in the CENP-A nucleosome impacted by protein-protein interactions with centromere protein N †

with a 12 Å cutoff and a grid spacing of 2 Å. Van der Waals interactions were treated with a 12 Å cutoff. Three sepa- rate replicas of each system were simulated for a duration of 10 µs by starting from different random seeds. The solvated CENP- N protein without the NCP was simulated for three independent replicas using the same protocol as for other complexes, exclud- ing the DNA relaxation step. The system specific information is specified in Table 1. SIRAH-Backmap tools 79 were used for backmapping CG-MD trajectories into atomistic resolution. The backmapping pro- cedure involves reconstructing atomistic positions on a per- residue basis 80, preserving the geometric structure (internal co- ordinates), followed by protonation and minimization using the atomistic force field ff14SB atomistic force field 81 in Amber- Tools82 tleap module. 2.3 Analysis of nucleosome dynamics To characterize the dynamical properties of the CENP-A nucleo- some and its structural changes upon the specific protein bind- ing simulated with CG-MD, we applied a set of analyses ex- plained here. Global stability was monitored through the root- mean-square deviation (RMSD), while local flexibility was cap- tured by root-mean-square fluctuations (RMSF). For both analy- ses, we considered the backbone CG beads of both histone and DNA. RMSD and RMSF were calculated relative to the energy- minimized structure explained in Section 2.2. Changes in the overall compaction of the nucleosome were quantified using the radius of gyration (Rg), which is a mass-weighted root-mean- square distance of all beads from the center-of-mass (COM). To probe sequence- and region-specific histone–DNA interactions, we computed contact maps between protein and DNA beads. Fol- lowing earlier work 83, native contacts were defined when the specified beads (GC for protein representing carbon atom, PX for DNA representing backbone phosphate atom) were within 7 Å. In the SIRAH CG representation, the direct identification of specific interactions such as hydrogen bonds or salt bridges is not feasible due to the reduced resolution of the model. In- stead, residue–residue interactions were characterized using this distance-based contact definition. For each residue pair, the av- erage contact population value was calculated as the fraction of simulation frames in which the contact was present. An average contact value of 1.0 indicates that the residues remained in con- tact throughout the entire trajectory , representing a highly sta- ble interaction. Contacts with values below 0.4 were classified as transient or weak, indicative of higher flexibility in that re- gion. The average contact values between protein residues were further visualized in the form of 2D contact maps, where the axes correspond to residue indices and the color scale indicates the stability of the contacts across the trajectory . A dark/intense color (the average contact value close to 1.0) shows that the two residues stayed in contact throughout the trajectory (stable, per- sistent interaction) while lighter color (less than 0.4) shows weak or transient contacts. All analyses were performed using GRO- MACS tools, while MDAnalysis was utilized to calculate the con- tact map 84. 4 | 1–16 + P V S O B M
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.CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint The secondary structure of proteins was calculated using SIRAH secondary structure tool. It uses the positions of CG backbone beads to approximate the backbone geometry defined as a structured element in terms of helical and extended re- gions (i.e. β-strands), or unstructured element defined as a coil. Since the SIRAH CG geometry of proteins retains enough infor- mation about local backbone curvature and spacing, these CG- based geometric rules provide an accurate distinction between helices from sheets 59. To extract dominant modes of motion in histone CENP-A upon CENP-N binding and to construct free en- ergy landscapes (FEL), representing thermodynamic map of con- formational space of a protein, the principal component analy- sis (PCA) was employed. It is capable to reveal the functionally relevant conformational states of molecules during the trajecto- ries and reduce dimensionality that helps in identifying configu- rational spaces with a limited number of degrees of freedom. In this method, a 3N × 3N covariance matrix of positional fluctua- tions of CG beads relative to every other coordinates over time is constructed based on simulated trajectory . Thus, it captures the correlated motions of coordinates, and the diagonalization of this matrix yields eigenvectors, representing principal motion direc- tions, and the corresponding eigenvalues, indicating the magni- tude of fluctuations along these directions. The trajectory is then projected onto these eigenvectors to derive the principal compo- nents (PCs). The first two PCs describe the dominant large-scale motions and are oftenemployed to construct a two-dimensional FEL. It is based on the estimation of the joint probability den- sity function (P(PC1,PC2)) obtained from a histogram of PCs and defined as ∆GFEL (x, y) = −kBT ln(P(x, y)/Pmax), where Pmax is the probability of the most probable state, kB is Boltzmann constant and T is the temperature. The PCA and FEL were calculated us- ing MDAnalysis Python library 84,85 providing input coordinates of CG beads. Together, the analyses performed provide a compre- hensive view of nucleosome stability , flexibility , as well as collec- tive dynamics. The errors for the quantities calculated were estimated where applicable. The error of the average is given by s/√ n, where s is the standard deviation of the mean values over the replicas, and n represents the number of replicas. 2.4 Umbrella Sampling simulations To understand PPIs and binding free energy between CENP-A pro- tein in an NCP and CENP-N CCAN protein, US simulations us- ing SIRAH FF were performed 62,86. The initial structure of the complex was taken from the final structure obtained from the atomistic simulations as discussed above. It was placed in a box of 320 × 200 × 160Å 3 , so that the complex was aligned parallel to the X-axis. The system was solvated, neutralized, and mini- mized as per the prior simulation protocol, explained in Section 2.2. Equilibration was then performed under NVT and NPT con- ditions, maintaining consistency with the MD simulation proto- col. To initiate the US simulation, at first we performed steered molecular dynamics (SMD) simulation to generate initial confor- mations for US windows. The CENP-N protein was pulled away from its initial position near the CENP-A side of the NCP into the bulk solvent along the X-axis, applying a harmonic pulling po- tential with a force constant of 1000 kJ mol−1 nm−2 with a pulling rate of 0.0001 nm/ps. To prevent drifting of the system along the reaction coordinate, a harmonic restraint with force constant of 20 kJ mol−1 nm−2 for whole nucleosome, except CENP-N protein, was applied. Positions of the molecular system simulated were saved during the course of pulling with 60 structures generated as US windows for separate MD simulations. Each window struc- ture was saved every 0.1 nm from the initial position of CENP-N and up to a COM distance between the CENP-A and CENP-N pro- teins of 7.95 nm. From a COM distance of 8.05 to 14.65 nm, the spacing was fixed at 0.2 nm. The schematic of US procedures is depicted in Fig. S1. Each system in the US window was independently further sim- ulated for 25 ns in thr NPT ensemble followed by an additional MD simulation for 50 ns with a V-rescale thermostat and Par- rinello–Rahman barostat. A harmonic bias potential with a force cosntant of 1000 kJ mol−1 nm−2 was applied to each window. This value was selected within the range (500–2000 kJ/mol· nm) re- ported in previous SIRAH umbrella sampling studies62,63 on pro- tein–protein interactions and was further optimized to achieve adequate histogram overlap between adjacent windows, ensur- ing sufficient sampling across the reaction coordinate. Fig. S2 depicts overlap of histograms from different windows obtained along reaction coordinate. Finally , the potential of mean force (PMF) was derived using the weighted histogram analysis method (WHAM) 87,88 as implemented in GROMACS to eliminate the in- fluence of the applied bias. Statistical errors were estimated by using default Bayesian bootstrapping algorithm built into the WHAM program 88. The binding free energy (∆G) between proteins, indicating the strength and the character of PPIs was calculated as the differ- ence in free energy between the bound and unbound states. The unbound state was defined as a conformational state where in- teractions between the proteins are completely diminished, cor- responding to a plateau (near-zero interaction) in the PMF plot. ∆G can be calculated mathematically as follows: ∆G = (−kBT ln Z bound e−Φi/kBT ) − (−kBT ln Z unbound e−Φi/kBT ) (1) Φi represents the PMF value associated with the ith bin along the reaction coordinate. The error in the binding energy is calcu- lated by propagating the errors89 from the minimum PMF (bound state) and the plateau region (unbound state) using the following equation: σ∆G = q σ 2 min + σ 2 plateau (2) Here σmin is the error at the minimum PMF and σplateau is the average error in the plateau region. 3 Results 3.1 Stability of CENP-A nucleosome upon CENP-N binding Since the function of NCPs depends on their structural flexibility , interactions with other NCPs or molecules, as well as upon chang- ing environment conditions, we aimed to analyze the stability of CENP-A nucleosomes in the presence and absence of CENP-N pro- + P V S O B M
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1–16 | 5 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint 180° (a) (b) (c) CENP-A Nucleosome CENP-A Nucleosome + CENP-N Protein Fig. 2 (a) The average root mean square deviation and (b) radius of gyration of the nucleosome with and without histone tails considered and the nucleosomal DNA alone. Structural parameters for the CENP-A nucleosome with and without CENP-N protein are marked in red and black, respectively. (c) The structure of the systems simulated after 10µs CG-MD simulations: the CENP-A nucleosome + CENP-N protein complex (top), and CENP-A nucleosome (bottom). Two views of the same complex are depicted. tein. For that, we calculated RMSD and Rg using data obtained for three independent replicas (see more details in Fig. S3 and Fig. S4). The RMSD has been calculated for both the histone protein core and nucleosomal DNA separately . We further cate- gorized the RMSD calculations for the histone core based on the inclusion and omission of the residues present in histone tails, which are generally inherently flexible, thus, hindering the un- derstanding of subtle changes in other protein regions. The re- spective data are labeled as "with tails" and "without tails" in Fig. 2. The time evolution of RMSD for all cases is presented in Fig. S3 while time dependent RMSD of only histone tails are depicted in Fig. S5 . The average RMSD plot (Fig. 2a) shows that considering hi- stone tails in the RMSD calculation, the fluctuation is higher in the system with bounded CENP-N to the CENP-A system (RMSD of 7.51±0.33 Å, see marked in red) as compared to the unbound CENP-A system (6.94±0.10 Å, in black). It is connected to the in- teractions induced by the presence of CENP-N explained further. The RMSD of systems without the consideration of the histone tails is lower, indicating higher histone core stability . In addition, the RMSD of the histone core in the presence of CENP-N is even slightly smaller, i.e., 5.12±0.08 Å while in the absence of CENP-N the average RMSD is 5.34± 0.08 Å. This indicates a possible sta- bilization of the nucleosome upon CENP-N binding. The average value of RMSD of nucleosomal DNA in the presence and absence of CENP-N is 5.45± 0.21 Å and 5.63± 0.08 Å, respectively (Fig. 2a). It suggests a slight rigification of some base pairs that are located near the CENP-N binding site, permitting their smaller structural deviations. Together with a slight histone core struc- tural stabilization, it provides evidence for possible NCP plasticity changes, which we discuss in the following section. The average radius of gyration for both systems is depicted in Fig. 2b. The presence of CENP-N protein does not alter larger structural moves and the shape of the NCP. Time evolution of Rg is shown in Fig. S4. The final structure of the NCPs after the sim- ulation is visualized in Fig. 2c. The upper panel depicts the CENP- A nucleosome with bound CENP-N, while the lower panel shows the unbounded CENP-A NCP. Distinct orientations are displayed for each system to convey the complete molecular architecture and assembly . The figure demonstrates that the systems consid- ered in this study retained their structural integrity throughout the microsecond-scale CG-MD simulations. 3.2 CENP-N binding interface: contacts with CENP-A and DNA CENP-N interacts specifically with the CENP-A histone and nu- cleosomal DNA, forming extensive contacts that are critical for its binding specificity and centromere function. Fig. 3a highlights the interaction interface between DNA, CENP-A, and CENP-N. The L1 loop of CENP-A is shown in red, while the DNA region, spanning SHL 2 to 3.5 (see Fig. 1c) that participates in binding, is high- lighted in yellow. Residues of CENP-N interacting with CENP-A 6 | 1–16 + P V S O B M
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.CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint SHL 2-3.5L1 loop CENP-N residueswith DNACENP-N residueswith CENP-A (a)(b) (d) (c) Fig. 3 (a) The binding interface involving residues from CENP-A, CENP-N, and DNA bases was obtained from the cryo-EM structure. Red indicates the L1 loop of CENP-A; yellow highlights DNA from SHL 2 to 3.5, the region interacting with CENP-N. Green denotes the CENP-N amino acids that interact with CENP-A, while blue marks those interacting with the DNA SHL. (b) Contact map showing interactions between CENP-N and CENP-A residues. (c) Contact map showing interactions between CENP-N residues and DNA bases. (d) Binding interface obtained from CG simulation with color coding as in panel (a). and DNA are shown in green and blue, respectively . This repre- sentation provides a structural overview of the binding sites iden- tified from experimental cryo-electron microscopy analysis 25. To evaluate the stability of experimentally observed residue contacts under dynamic conditions, we analyzed these interactions over 10 µs of CG-MD simulations, from which 8 µs were considered for data analysis. In Fig. 3b, the contact map between CENP-N and CENP-A residues over 4000 snapshots from CG-MD simulations is de- picted. This contact analysis (see Methods section for de- tails) confirms that CENP-A residues forming the RG loop (i.e., ARG80–GLY81) interact with CENP-N, consistent with cryo-EM data, and suggests stable contacts at this interface during CG- MD. ARG80 of CENP-A interacts strongly with residue ASN145 of CENP-N, resulting in the average contact value of 0.79. Its inter- action with residues PRO144 and GLN146 from CENP-N is weaker (contact value of 0.21 and 0.11 respectively), indicating a broader contact region. Residue GLY81 stands out with strong and mul- tiple contacts, notably with residues ASN145 and GLN146 (both with an average contact population of 1.00), and significant inter- actions with residues PHE8, PRO144, and TYR147, pointing to a highly engaged interface. The average contact population values of all participating residue pairs are listed in Table S2. To maintain its main function, CENP-N should primarily bind to the CENP-A protein in the histone core23,90. However, it addition- ally shows the interaction with the DNA as depicted in Fig. 3a, therefore, the respective contact map between the CENP-N pro- tein and the DNA was calculated (see Fig. 3c). The CG-MD simu- lations denote several persistent contacts between the DNA bases and CENP-N residues. DNA base G98 exhibits strong interactions + P V S O B M
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1–16 | 7 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint 180° (a) (b) ΔRMSF >0 ΔRMSF <0 1 1 2 3 2 (c) (d) Fig. 4 Root mean square fluctuations of the CENP-A protein in the presence (red) and absence (black) of the CENP-N protein. RMSF values were averaged over three independent replicas for both systems. The cyan-shaded areas denote CENP-A residues that exhibit higher RMSF in the presence of CENP-N, while the green-shaded areas mark residues with lower RMSF. These residues are further highlighted in (b), where the final CG-MD structure is depicted with the color coding consistent with the RMSF plot. The free energy landscape of CENP-A along PC1 and PC2 shown in the presence (c) and absence (d) of CENP-N. Representative structures of CENP-A corresponding to each minimum are depicted in panels outside the figure. ∆GFEL is provided in kcal/mol. with ARG169 and LEU168 of CENP-N, with average contact pop- ulation of 0.99 and 0.73, respectively . The high frequency of con- tact formation indicates the attractive character and stability of the CENP-N binding at this interface (see Table S3). Similarly , G99 exhibits stable contacts with LYS81 and VAL82, both showing maximum contact values of 1.00, along with a weaker interaction with TRP83. The adjacent base A100 also forms strong contacts, particularly with LYS148 (1.00), TYR147 (0.85), and again with LYS81 (0.97), indicating a consistent role of this region in DNA recognition. A43 base from DNA strand-2 (DNA base number 190 in Fig.3c) strongly interacts with ARG44, LYS45, and GLU46, with contact values exceeding 0.75, while C44 (DNA base num- ber 191 in Fig.3c) forms multiple contacts with MET18, ASN19, and LYS45, further supporting weak interaction with ARG44. T49 ((DNA base number 196 in Fig.3c)) also shows a modest contact with ASN19. These interactions (see values listed in Table S3) highlight key regions of CENP-N that stably associate with the DNA, suggesting their role in mediating nucleosome binding and stabilizing the DNA–protein interface in the CENP-A nucleosome. The binding interface formed by CENP-A, CENP-N, and DNA is depicted in Fig. 3d, which represents the final snapshot taken af- ter 10 µs-long MD simulation. The amino acid residues from the proteins as well as the DNA nucleic bases are shown in surface representation, using the same color scheme as described in Fig. 3a. 3.3 CENP-A structural stability upon CENP-N binding The CG-MD simulations indicate that the overall structure of the CENP-A-containing NCP remains largely unchanged upon CENP-N binding (Fig. 2). To examine potential structural changes specif- ically in the CENP-A protein within the histone core in the pres- ence and absence of CENP-N, the RMSF of CENP-A was calculated (see Fig. 4a). The region near the L1 loop of CENP-A (specifically residues CYS75, VAL76, LYS77, PHE78, THR79, ARG80, GLY81, VAL82) possesses lower fluctuations in the CENP-N bound state (data in red) compared to the unbound state (data in black), in- dicating that CENP-N binding stabilizes this region. Fluctuations in the C-terminal residues (Res 134-Res 140) remain largely un- changed. However, the N-terminal residues exhibit higher value of RMSF in the presence of CENP-N (exceeding 1 Å). Such an observation supports that CENP-N binding induces increased flex- ibility at the N-terminal region, potentially facilitating conforma- tional adjustments required for further stability . We marked the CENP-A residues showing higher and lower RMSF in the pres- ence of CENP-N relative to the CENP-N unbound state in cyan and green, respectively . These residues are shown in Fig. 4b (with re- spective regions indicated in Fig. 4a), which represents the final snapshot taken after 10 µs-long MD simulation. 8 | 1–16 + P V S O B M
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.CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint Table 2 Secondary structure content (in %) for CENP-N protein regions in bound and free states. Errors are indicated as mean± error. Res 1-50 Res 51-200 Res 201-295 Category Bound Free Bound Free Bound Free Stable ss: Helix 52.67± 3,75 49.37± 2.37 30.67± 1.01 32.13± 1.67 30.60± 0.99 33.07± 2.20 Stable ss: Extended 5.03± 0.88 4.70± 2.36 23.07± 1.90 22.30± 1.22 8.20± 1.56 5.73± 0.95 Stable ss: Helix + Extended 57.70± 3.85 54.07± 3.45 53.74± 2.16 54.43± 2.06 38.80± 1.84 38.80± 2.40 Coil 42.30±3.96 45.97± 1.60 46.23± 1.31 45.57± 1.19 61.17± 0.67 61.17± 1.80 To further understand the CENP-A conformational change upon the CENP-N binding, we performed the PCA analysis as described in the Methods section. In Fig. 4c,d, the FEL plots along two principal components (PC1 and PC2) for CENP-A histone in the presence as well as absence of CENP-N are visualized. In both cases, we observe distinct energy basins indicating distinct stable conformational states of CENP-A. In the absense of CENP-N, the two stable conformational states of CENP-A are structurally sim- ilar and are separated by the low energy barrier. However, the number of basins upon the CENP-N binding increases (Fig. 4c), representing stronger structural changes. Such conformational changes likely reflect the specific interactions between CENP-A and CENP-N, stabilizing additional conformational states and re- vealing a more complex free energy landscape. The identified rep- resentative structures corresponding to each energy basin for both cases are depicted in panels outside in Fig. 4c,d. They were iden- tified by picking conformations at the minima of the FEL spanned by PC1 and PC2 shown in Fig. 4c,d. We used backmapped all- atom structures to better visualize the conformational changes across the different basins. We see that the stable helical regions remain largely unchanged between structures, while the loop re- gions exhibit distinct conformations in each minimum. In the CENP-N unbound case (Fig. 4d), the representative structures of CENP-A also show variations in loop conformations while main- taining the helical structure. 3.4 Free and bound states of CENP-N To explore whether CENP-N and CENP-A coevolve to support NCP’s function in the centromere region, we analyzed the struc- tural dynamics of CENP-N both in its complex with the CENP- A-containing nucleosome (labeled as bound CENP-N) and in its isolated state (labeled as free CENP-N). Hence, we performed an additional CG-MD simulation of free CENP-N as explained in the Methods section. Fig. 5 shows the set of analyses conducted to demonstrate the structural differences. Using RMSF calcula- tion depicted in Fig. 5a, differences in fluctuations of specific regions of CENP-N in its both states are visualized. The RMSF plot clearly demonstrates that residues 1–200 become more or- dered (stabilized) upon nucleosome binding (see data in red), whereas residues 201–295 display consistently higher RMSF val- ues in both states. Thus, in the bound case, CENP-N exhibits in- creased flexibility in its C-terminal residues res216-res255 (cyan colored region in Fig. 5a) which are known to be disordered according to previous experimental studies 30. In contrast, the N-terminal residues display lower RMSF values in the bounded state of CENP-N, indicating significant stabilization due to interac- tions with CENP-A and nucleosomal DNA. The N- and C-terminal residues are highlighted in the final conformation of CENP-N in both the bound and free states in panels outside Fig. 5a. Both the CG representation, as well as the backmapped all-atom structures of the protein are visualized. In the CG representation, the CG beads of each amino acid are colored according to their RMSF values as indicated in the color bar. Regions of the molecule with relatively higher RMSF values, indicating greater flexibil- ity , are shown in red, while regions representing relatively lower fluctuations are shown in blue. The RMSF coloring of CENP-N was generated using a user-defined color scale in VMD 91. This coloring reflects relative differences in flexibility across the pro- tein rather than absolute RMSF values. The N- and C-terminal residues are marked within dashes circles in the CG representa- tion, while they are highlighted in pink over the protein’s sec- ondary structure in the atomistic representation. The N-terminal residues 1–50 of CENP-N show a more intense red color of the CG beads in the free state of the protein, indicating their higher fluctuations. In the bound state, these fluctuations are signifi- cantly reduced, showing medium relative RMSF. In contrast, the C-terminal residues (201–295) display similar color patterns in both free and bound states, suggesting comparable fluctuations, which result in a higher flexibility of this region present in both states of CENP-N. The backmapped all-atom structures reveal that several residues from the N-terminus of CENP-N adopt loop-like confor- mations in both states, despite largely belonging to helical re- gions. By contrast, the C-terminal tail predominantly forms loop structures in both bound and free states. Henceforth, it is im- portant to calculate the secondary structure (ss) elements of the CENP-N protein in both the bound and free states across MD sim- ulation replicas. At first, we performed the ss analysis for protein at both free and bound state using SIRAH ss tool as explained in

section. Fig. 5b shows the average ss content, along with error bars from three independent replicas, for both sys- tems. We also calculated the time evolution of the ss content for the bound and free states of CENP-N (see Fig. S6) over the last 8 µs. Both results revealed no significant changes in the ss between the two states. Considering the differing flexibility of particular regions of the protein, we identified three different re- gions that were studied separately in more detail. The first region was located between residue 1 to 50, which showed the strongest changes in the RMSF behavior upon binding. The second region (residue 51 to 200), demonstrating no significant RMSF changes upon binding and the third region (residue 201 to 295), which re- main disordered indicating higher RMSF in both bound and free state (Fig. 5a). The secondary structure for these three differ- ent regions is given in Table 2, where helix and extended regions represent stable ss, while coil represents unstructured ss. From these data, we see that the first region shows increased contri- + P V S O B M
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1–16 | 9 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint (a) (b) (c) Bound CENP-N Bound CENP-N Free CENP-N Free CENP-N RMSF of GC bead Fig. 5 (a) Root mean square fluctuations of the bound (red) and free (black) CENP-N protein, averaged over three independent replicas. . The cyan-shaded areas denote CENP-N residues that exhibit higher RMSF in bound state, while the green-shaded areas mark residues with lower RMSF with respect to free state. Final structures of both free and bound CENP-N are shown at CG and backmapped all-atom resolutions. In all-atom representation, the N- or C-terminal residues are highlighted in pink, while the rest of the protein is shown in yellow. b) Percentage of secondary structure elements observed over the simulation trajectory for both bound and free CENP-N. (c) Average RMSD plots of unbound and free CENP-N for three regions: overall, residues 1–200, and residues 201–295. Error bars represent standard deviations from three independent replicas. bution of stable ss from 54.07% to 57.70% (especially for helix content, which changes from 49.37% to 52.67%) upon binding as compared to the free state of the protein. In the other regions, the stable ss content versus coil does not change markedly: less than 1% of stable ss decreases or remains unchanged, with over- all flexibility largely unaffected upon binding with CENP-A and nucleosomal DNA. Since experimental studies, such as HX exchange 30, have re- ported that the N-terminal domain of CENP-N adopts a folded conformation upon interaction with CENP-A, particularly involv- ing the first 200 residues, we examined the combined dynamics of the ordered region (residues 1–200) and the disordered re- gion (residues 201–295) in CENP-N through RMSD calculation. At first, we calculated the RMSD of whole CENP-N in its free and bound states, see Fig. 5c. The overall RMSD was significantly higher for free CENP-N than compared to the bound CENP-N with the average RMSD of 14.32±0.44 Å and 9.98±0.93Å respectively . At the same time, ordered residues (res 1-200) show a decrease in RMSD from 11.18± 0.45Å to 5.57± 0.22 Å upon nucleosome binding, whereas the flexibility of the disordered region remains similar in both conditions. To further assess the compactness of CENP-N considering residues in these different regions, we calcu- lated the corresponding Rg (see Fig. S7). Rg is slightly smaller in the bound state of CENP-N, however, the average Rg for all three cases (entire CENP-N, the ordered region, and the disor- dered region), indicates less differences than the average RMSD. Thus, while region-specific RMSD analysis reveals local stabiliza- tion of the N-terminal residues upon nucleosome binding, the overall Rg remains largely similar, indicating that CENP-N retains its global size and shape while undergoing local conformational adjustments. 3.5 CENP-N binding free energy To quantify the strength of the PPIs between the CENP-A pro- tein in the NCP and the modulating CENP-N protein, the binding energy was calculated using the US method. Here, we consid- 10 | 1–16 + P V S O B M
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.CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint 180° ξ = 8.0 nm Bound ξ = 5.45 nm 180° Unbound ξ = 14.45 nm (a) (b) (c) (d) (a) Fig. 6 (a) The PMF curve representing the binding free energy between the CENP-A and CENP-N proteins in the centromere NCP obtained using the umbrella sampling method. The error bars calculated using Bayesian bootstrapping algorithm are depicted in grey. The 3D representation of the binding site and the proteins involved is depicted using CG beads, along with the nucleosomal DNA depicted in white surface representation. Different structures representing the COM reaction coordinate,ξ, of: (b)ξ = 5.45 nm, (c)ξ = 8.0 nm, and (d)ξ = 14.45 nm, are visualized for clarity. Bead colors represent the relative RMSF fluctuations, where red beads indicate regions of CENP-A and CENP-N with the highest fluctuations, while blue beads indicate the smallest fluctuations. The RMSF coloring was generated using a user-defined color scale in VMD91. Two views of the same system are depicted at the selected COM reaction coordinate. ered the reaction coordinate, ξ, as the COM distance between the CENP-A protein and the COM of the ordered part of CENP-N (res 1-200). The choice of the ordered region of CENP-N was guided by the RMSF and RMSD analyses depicted in Fig. 5a,c, which showed that residues 201–295 remain disordered irrespective of nucleosome binding. Focusing on the ordered regions ensures that the sampled conformational space reflects meaningful inter- actions and avoids contributions from highly flexible segments. The resulting PMF plot along with the calculated error bars is presented in Fig. 6a. The binding energy of the complex can be calculated from the difference between the highest and lowest values of the average PMF curve (details in the Method section). Thus, the binding free energy between CENP-A and CENP-N is concluded to be -7.92±0.99 kcal/mol. To visualize the structural changes of CENP-A and CENP-N at three different ξ values, the representative structures of the sys- tem calculated were further inspected. To do so, the CG beads of all residues in both proteins were visualized and colored ac- cording to their relative RMSF values, calculated from the re- strained trajectories at the corresponding ξ value. A more in- tense red color indicates significant fluctuations of molecular en- tity representing the bead, while a more intense blue color rep- resents smaller fluctuations. At ξ = 5.45 nm, corresponding to the bound state between the proteins, we observe higher fluctu- ations in the disordered region of CENP-N that is not involved in the direct protein-protein contact (Fig. 6b). At ξ = 8.0 nm, fluc- tuations at both terminals of CENP-N increase (Fig. 6c), while in the unbound state (ξ = 14.45 nm) they display much stronger fluctuations (Fig. 6d). The unbound state of CENP-N essentially represents the free state of CENP-N, where a substantial portion of the protein exhibits a disordered nature. For CENP-A, fluctua- tions increase in the unbound state compared to the bound state, although the increase is less significant compared to CENP-N since the protein is bounded inside the histone core. The higher struc- tural stability of both proteins upon their binding is clearly visible from this analysis and complements the dependencies observed in the unbiased MD simulations. 4 Discussion The plasticity of the centromeric NCP is a key factor in facilitat- ing its function 92. Its interaction with binding proteins directs chromatin folding and the compaction of genetic information, processes that are essential for accurate chromosome segregation and the maintenance of genome stability 11. Here, we examine the binding of CENP-N and the resulting structural changes in the NCP. Overall, our study investigates the PPIs within the NCP, with + P V S O B M
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1–16 | 11 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint a particular focus on how CENP-N binding influences the dynam- ics and stability of the CENP-A nucleosome. Because nucleosome dynamics occur over extended timescales, we employed a CG-MD to capture these interactions and evaluate how the underlying protein–protein associations contribute to nucleosome function- ality . The RMSD analysis of NCPs without histone tails reveals no significant structural differences upon the CENP-N binding with rather small structural stabilization of 0.22 Å (Fig. 2a), indi- cating that the core histone fold remains structurally stable ir- respective of the CENP-N presence. However, when histone tails are included in this analysis, a modest increase in RMSD in the presence of CENP-N is observed, suggesting that the histone tails may adopt more dynamic or altered conformations upon CENP- N binding. Interestingly , the radius of gyration remains largely unchanged across all comparisons, including those performed for histone cores with and without tails and the DNA (Fig. 2b). This indicates that despite local flexibility changes, particularly in the histone tails, the overall compactness of the nucleosome remains constant. These results suggest that the role of the CENP-N pro- tein is not to further compact individual nucleosomes, but rather to facilitate inter-nucleosomal interactions that were reported to drive centromeric chromatin folding and organization 29. Starting with the binding interface involving DNA bases, CENP- A amino acids and CENP-N amino acids reported in cryo-EM 29, we performed several replica MD simulations each of 10 µs, which revealed stable residue-residue contacts between the NCP and CENP-N. PPIs between CENP-A and CENP-N were shown to demonstrate a high specificity of the interaction interface en- riched in polar, charged, and aromatic side chains, suggesting diverse binding interactions. The RG loop of CENP-A within its L1 loop, ARG80 and GLY81 emerge as central points of contact in the simulation based on the average contact values (see Tab. S2), which were also suggested by the cryo-EM study of Chit- tori et al. 25. Considering the chemical nature of the individ- ual contributing residues, we found that ARG80 of CENP-A, with its positively charged guanidinium group, interacts strongly with ASN145 of CENP-N and moderately with PRO144 and GLN146. These residues possess polar or partially polar side chains, en- abling electrostatic and hydrogen bond-based interactions. Even if the SIRAH CG FF cannot properly treat subtle hydrogen bonds, it is known to adequately represent electrostatic interactions, which are essential in proper mimicking the PPIs of the complex studied. Notably , GLY81 of CENP-A exhibits a high degree of flexi- bility and forms extensive contacts with ASN145, GLN146, PHE8, PRO144, and TYR147 of CENP-N, supporting the idea that this position serves as a hub for both polar and hydrophobic inter- actions. THR79, a polar uncharged residue of CENP-A, interacts strongly with THR4 of CENP-N, with an average contact value of 0.99 (see Table S2), likely forming a hydrogen-bond network via their side-chain hydroxyl groups. Additionally , a hydropho- bic VAL82 participates in both polar (ASN145, GLN146) and aro- matic (PHE8, TYR147) contacts with CENP-N, suggesting van der Waals and hydrophobic interactions may also contribute. More- over, a negatively charged ASP83 forms notable contacts with polar aromatic TYR147 (average contact value 0.55), which are likely mediated through hydrogen bonding or electrostatic attrac- tion. These findings indicate that the binding interface between CENP-A and CENP-N is stabilized through a combination of di- verse interactions, which together contribute to a high PPIs speci- ficity and strength of the CENP-A/CENP-N interaction, which was calculated to be -7.92±0.99 kcal/mol based on umbrella sam- pling simulations performed in this work. Interestingly , while cryo-EM studies25 have previously identified GLU3, THR7 as par- ticipating in the interaction interface between CENP-N and the RG loop of CENP-A, the contact-based analysis performed based on CG-MD simulations in the present work did not reveal stable or significant interactions involving these residues. The interaction between the nucleosomal DNA and CENP-N was also probed using a distance cutoff approach. The DNA bases, primarily belonging to SHLs 2 to 3.5, and amino acids from CENP- N involved in the interaction are listed in Table S3. This obser- vation is consistent with previous experimental studies 25. The simulation suggests that several arginine and lysine residues from CENP-N, such as ARG169, LYS81, LYS148, ARG44, and LYS45, in- teract with DNA, indicating that the interaction is predominantly electrostatic. The distance-based interaction analysis also identi- fied additional amino acids likely to participate in DNA binding. For example, LEU168 and VAL82 were detected to interact with DNA through hydrophobic contacts, while GLU46 and TYR147 frequently contact DNA bases (average contact value 0.76 and 0.85 respectively), suggesting potential hydrogen-bond-like inter- actions in the coarse-grained representation. MET18 and ASN19 are also recognized as potential DNA binding residues due to their ability to form hydrophobic interactions and hydrogen bonds, re- spectively . To monitor any allosteric changes in the histone core upon binding of CENP-N, we focused on the conformational arrange- ment of the CENP-A protein in the presence and absence of CENP- N. The RMSF analysis of CENP-A in Fig. 4a revealed sequence- dependent differences in the structural flexibility upon CENP-N binding. The L1 loop region of CENP-A exhibits reduced RMSF in the presence of CENP-N, whereas in the absence of CENP-N, this region displays markedly increased flexibility . This suggests that binding of CENP-N confers structural stabilization to the L1 loop, potentially through direct interactions. RMSF analysis fur- ther shows that the N-terminal region of CENP-A exhibits higher flexibility when bound to CENP-N compared to its unbound state. This suggests that interaction with CENP-N enhances the dynamic behavior of the CENP-A N-terminus. In parallel, contact map base analysis (Fig. S8) revealed a reduction in the average number of contacts formed by residues 13–22 of CENP-A with histone H2A when CENP-N is present (Fig. S8a). Conversely , in the absence of CENP-N, this region engages in more persistent contacts with H2A (Fig. S8c) with participating residues marked within a circle. Overall, this study indicates that the binding of CENP-N alloster- ically disrupts contacts between the CENP-A N-terminal tail and histone H2A, thereby enhancing the conformational freedom of the N-terminus. The enhanced fluctuations of the CENP-A N-terminal tail (see Fig. 4a) observed in our simulations suggest that this region relies on interactions with specific binding partners for protein stabiliza- 12 | 1–16 + P V S O B M
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.CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint tion when bound to CENP-N. This dynamic behavior is consistent with experimental observations93, highlighting the functional im- portance of the N-terminus of CENP-A for centromere functional- ity and kinetochore assembly . For example, the N-terminus of CENP-A was shown to contribute to the stabilization of the cen- tromere binding protein CENP-B by direct interaction94,95. More- over, it acts as a recruiter of key kinetochore proteins such as CENP-C and CENP-T at both ectopic sites and endogenous cen- tromeres in Schizosaccharomyces pombe and human cells 96,97. Thus, the enhanced fluctuations of the N-terminal tails of CENP- A, as captured by RMSF in Fig. 4a, may serve as a signature of its active engagement in modulating interactions essential for cen- tromere function. Furthermore, CENP-A’s unstructured N-terminal tail bears post- translational modifications98. Several studies have demonstrated that for instance phosphorylation of serine 7, 16 or 18 within the CENP-A N-terminal domain significantly influences centromeric chromatin structure and function 99–101. These residues lie near the region that shows altered contact behavior in CG simulations (see residue 13 to 22 in Fig. S8), suggesting that CENP-N binding may modulate the exposure or accessibility of phosphorylation sites, thus indirectly impacting downstream chromatin remodel- ing or signaling events (see Fig. S8 a). Therefore, the observed destabilization of local contacts (Fig. S8 a) and increased flexi- bility of the N-terminus of CENP-A (residue 13 to 22 in Fig. 4a) upon CENP-N binding is not only structurally plausible but also bi- ologically meaningful, potentially contributing to regulating cen- tromere function via a modulation of post-translational modifica- tion, recruitment of kinetochore proteins, or higher-order chro- matin structure formation. To understand the molecular determinants of centromere as- sembly , it is essential to quantify the binding energetics of the CENP-A–CENP-N complex. Binding energy not only reflects over- all stability but also reveals how different regions of a multiva- lent protein contribute to interaction, providing mechanistic in- sight that cannot be captured by static structural measurements alone. Although, calculating binding energy experimentally for this nucleosome-like system is challenging to date due to the mul- tivalent and context-dependent nature of their interactions. Finally , the calculated binding free energy between the CENP-A- containing NCP and the CENP-N protein from CG-MD simulations in our work captures the strength and complexity of the interac- tion (Fig. 6a), while further analyses of trajectories generated uncovers the split nature of CENP-N: the N-terminal domain be- comes ordered upon binding to the NCP, whereas the C-terminal region remains largely disordered (Fig. 6b-d). A similar observa- tion was also reflected during the independent CG-MD simulation of CENP-N in its bonded and a free state as discussed in Section 3.4. RMSF base study supports the split nature of CENP-N (see Fig. 5a), where N- and C-terminals of CENP-N remain disordered in the free state, while the N-terminal becomes ordered in the bound state with the NCP. Such a localized structural ordering in our simulations aligns well with experimental HX exchange data that analyzed the structural dynamics of the N-terminal do- main of CENP-N bound to CENP-A nucleosome, as well as in its free state30. The experimental study reported substantial HX pro- tection throughout the N-terminal with up to 200 residues upon binding to the CENP-A nucleosome, indicating a transition to a more stable and ordered conformation. In our work, MD simula- tions show that the C-terminal region of CENP-N (Res 201-295) remains largely disordered, consistent with the experimental ob- servation that residues 209–240 are not involved in binding 25. Notably , the increased order in the bound state in our simulations does not involve major changes in the secondary structure (Fig. 5b) for the whole protein, with no global alterations observed over multiple replicas. However, regional differences are evident: the N-terminal segment (residues 1–50) exhibits increased stable secondary structure upon binding, suggesting its role in mediat- ing interactions with CENP-A and nucleosomal DNA. The mid- dle segment (residues 51–200) maintains a balanced distribution of structured and unstructured elements (see Table 2, showing minimal response to binding. By contrast, the C-terminal region (residues 201–295) is predominantly disordered in both states, consistent with its reported higher flexibility . Despite the local changes described, the overall Rg of CENP-N remains largely un- changed (a decrease of around 1.07± 0.86 Å was obtained, see Fig. S7), indicating that the global size and shape of the pro- tein are mostly maintained. Taken together, these findings indi- cate that binding of CENP-N to the NCP primarily stabilizes the N-terminal region of CENP-N, while the central and C-terminal regions remain largely unaffected. This study suggests that the binding-induced conformational changes in CENP-N are driven by localized ordering rather than large-scale structural rearrange- ments. 5 Conclusions In this study , we provide molecular insights into the interac- tion between the CENP-A nucleosome and one of its key bind- ing partners, CENP-N, using a combination of biased and unbi- ased molecular dynamics simulations. Notably , the binding in- terface predicted by cryo-EM studies remains conserved in the coarse-grained simulations performed, linking structural and dy- namical perspectives of this biological complex. In addition, CENP-N association enhances conformational fluctuations in the N-terminal region of CENP-A, a feature likely to facilitate fur- ther protein–protein interactions essential for centromere assem- bly and function. A split nature of CENP-N was observed in unbi- ased MD simulations, as well as upon binding to the nucleosome core particle revealed by umbrella sampling method. The multi- tude of analyses and dependencies reported in this work supports diverse experimental observations, thus demonstrating the SIRAH coarse-grained force field reliably captures the long microsecond- scale dynamics of this centromeric complex. Finally , binding free energy analyses provided a quantitative perspective on CENP-A–CENP-N association in NCPs, revealing in- sights into promising directions for the regulation of centromere function through post-translational modifications, chromatin re- modelers, or the rational engineering of synthetic centromeres. Together, our findings provide a critical mechanistic understand- ing in the characterization of protein-protein and protein-DNA in- teractions, deepening our perception of microscopic processes in centromeres and offering a foundation for future studies aimed at + P V S O B M
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1–16 | 13 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 10, 2025. ; https://doi.org/10.1101/2025.11.10.686994doi: bioRxiv preprint targeted control of centromere functionality . Subsequent studies may further explore experimental measurements of binding ener- getics to complement and validate the computational predictions presented here. Author contributions Abhik Ghosh Moulick: Writing – review and editing, Writing – original draft, Visualization, Validation, Software, Methodology , Investigation, Formal analysis, Data curation, Conceptualization. Sylvia Erhardt: Writing – review and editing, Validation, Funding acquisition. Wolfgang Wenzel: Writing – review and editing, Re- sources, Funding acquisition. Mariana Kozlowska: Writing – re- view and editing, Supervision, Resources, Project administration, Methodology , Funding acquisition, Formal analysis, Conceptual- ization. Conflicts of interest The authors declare no conflicts of interest. Data availability Data generated is available from the corresponding author upon reasonable request.

This research was made possible by funding from the Carl-Zeiss- Stiftung and Center SynGen. The authors gratefully acknowl- edge the computing time provided on the high-performance com- puter HoreKa by the National High-Performance Computing Cen- ter at KIT (NHR@KIT). This center is jointly supported by the Federal Ministry of Education and Research and the Ministry of Science, Research and the Arts of Baden-Württemberg, as part of the National High-Performance Computing (NHR) joint funding program (https://www.nhr-verein.de/en/our-partners). HoreKa is partly funded by the German Research Foundation (DFG).

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