Effect of Hydroxyapatite Surface on BMP-2 ... - ACS Publications

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The Effect of Hydroxyapatite Surface on BMP-2 Biological Properties by Docking and Molecular Simulation Approach Haojie Gu, Zhiyu Xue, Menghao Wang, Mingli Yang, Kefeng Wang, and Dingguo Xu J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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The Journal of Physical Chemistry

The Effect of Hydroxyapatite Surface on BMP-2 Biological Properties by Docking and Molecular Simulation Approach Haojie Gu†, Zhiyu Xue†, Menghao Wang#, Mingli Yang‡,£, Kefeng Wang§, ‡,* and Dingguo Xu†,‡* †College

of Chemistry, MOE Key Laboratory of Green Chemistry and Technology, and ‡Genome Research Center of Biomaterial, Sichuan University, Chengdu, Sichuan 610064, P. R. China.

§National

Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610065, P. R. China.

#Key

Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, P. R. China. £Institute

of Atomic and Molecular Physics, MOE Key Laboratory of High Energy Density Physics and Technology, Sichuan University, Chengdu, Sichuan 610065, P. R. China.

*Corresponding authors: KF Wang, [email protected] DG Xu,

[email protected]

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Abstract: The interactions between osteogenic proteins and biomaterials surface is crucial to the application of biomaterials, in which the conformational or orientational change of the adsorbed protein on the solid surfaces is one of the most important interactions other than the protein adsorption. Although some progress has been made in the mechanism of proteins adsorption on the surface of Hydroxyapatite (HAP) in recent years, there is still insufficient atomistic/molecular information about the conformation and orientation of protein upon adsorbing on solid surfaces. In this study, different orientations and conformations of Bone Morphological Protein- 2 (BMP-2) adsorbed on the surface of HAP were calculated through the protein-solid surface docking approach; the relationship between optimal adsorption and biological activity of BMP-2 was investigated by applying a in combination with Molecular Dynamic simulation (MD) and Steered Molecular Dynamic simulation (SMD). Two favorite adsorption conformers were screened out according to the docking results on the basis of orientations of BMP-2 with different epitopes. Subsequent MD and SMD results showed that the knuckle epitope of BMP-2 was easier to adsorb on the surface of HAP (100) than the wrist epitope companying with certain conformational changes. Such absorption mode led to the wrist epitope of BMP-2 being exposed to the environment. This can be specifically identified/interacted with type I receptors on the stem cell membrane, which further induces the differentiation of stem cells into osteoblasts. Current simulation provided a theoretical high-throughput screening method for the protein-biomaterials adsorption states. It can be extended to more researches about different proteins adsorption on the surface of different materials. The simulation results provided more information at the molecular and atomic level to further interpret the mechanism of osteoinductivity from the perspective of growth factor adsorption. Meanwhile, we believe it should be a meaningful attempt to screen biomaterials key factors by high-throughput method, which might become a promising way to develop or optimize new biomaterials.

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1. Introduction The interaction of protein and biomaterials surfaces is the key to design biomaterials and bioinstruments. The behaviors of proteins or cells are just based on the complex phenomenon of biomolecular interaction with the surfaces. When biomaterials are implanted in to living body, proteins play an important role in the determination of biological properties. Proteins are not only the intermediary to study the interactions between cells and material surfaces, but also effectively enhance the proliferation and differentiation of cells by adsorption of target protein molecules onto the material surfaces. From the perspective of protein, biomaterials achieve its biological properties mainly through two aspects. One is the adsorption of certain efficient functional proteins.1-2 Another is the conformational change of the adsorbed target protein due to the specific biomaterials’ properties.3-5 The adsorption of protein not only ensures that the proteins have suitable orientations, but also promises that the proteins maintain their active conformations. It directly affects the sensitivity and effectiveness of the interaction between proteins and cells. Many works on protein adsorption behaviors have been carried out to better understand the mechanism of protein adsorptions and the relationship between biomaterials and its biological properties. It has been attracted lots of attentions in the process of bone tissue repairing, especially, in the early stage of bone mineralization. Born Morphogenetic Protein-2 (BMP-2), which belongs to the protein of the transforming growth factors- (TGF-), is one of the most important bone regeneration proteins and usually applied as the target protein of research. It can induce the formation and even take part in the growth, development and reconstruction of the bone and cartilage.6-9 BMP-2 binding to its SER-THR receptors triggers a signal cascade of osteoblast differentiation that induces the formation of bone and participates in the growth, development and reconstruction.10-15 A large number of experiments have shown that there is high-affinity interactions between BMP-2 and active type I receptor, and the affinity binding to type II receptor is relative lower.13, 15-16 Hydroxyapatite (HAP), Ca10(PO4)6(OH)2, one of the calcium phosphates, is the major mineral component of the human bone.17-19 It has been widely applied in bone regeneration for its good biocompatibility, bioactivity, even osteoinductivity which is proved by Zhang et al.20-23 Due to the advantages of HAP, many researchers have 3



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explored the interactions between its surfaces and proteins, especially with BMP-2.9, 24-29

For instance, Long et al. confirmed the helical structure of the salivary protein

terminal adsorbed on HAP surface by ssNMR.30 Dolatshahi-Pirouz et al studied the adsorption behaviors of fibronectin on surfaces of HAP.31 Zhang et al explored the adsorption of chitosan molecules on different crystal planes of HAP by MD simulations.32 These studies revealed that the conformation, binding sites and activity of protein could be affected by the surfaces. In addition, some studies showed that the osteoinductivity of HAP might be caused by the specific adsorption of BMP-2 and changes of the conformation of BMP-2.9, 28-29 A suitable conformation makes BMP-2 to interact naturally with its receptors on the cell membrane, thereby triggering a signal to induce cell differentiation. For now, there are lots of researches about the adsorption of BMP-2 but the study of conformational changes of BMP-2 caused by the surfaces of HAP is still rare.33-37 Importantly, monomer BMP-2 molecule, which is inactive in reality, was usually adopted in previous simulations.9, 25, 38-39 Thus, in order to better understand the bioactivity and osteoinduction mechanism of HAP through the view of BMP-2, further investigations are then required to reveal the effect of material surface properties on the conformation of BMP-2 homodimer. Although a few of experiments have been conducted to study the conformation changes of BMP-2 on different surfaces,32,

40-41

the information at the

atomistic/molecular level is still inadequate. Computer simulation has become an important means of materials genome research,42 which gives new protocols for studying the interactions between proteins and material surfaces from micro perspectives. It has been adopted to investigate the adsorption of proteins on material surfaces, for example, Wu and coworkers studied the micro-adsorption mechanism of the 10th type III module of fibronectin (FN-III10), BMP-2 and BMP-7 on the surface of HAP by using molecular dynamics (MD) and steered molecule dynamics (SMD).9, 43-44

Utesch et al simulated the initial adsorption process of BMP-2 on hydrophobic

graphite and hydrophilic titanium dioxide.26 Zhou et al studied the adsorption orientation of FN-III10 and FN-III7-10 adsorbed on HAP (001) surfaces in cavities of calcium ions with Parallel tempering Monte Carlo (PTMC) and MD simulation methods.45 Similar with above studies, most of researchers usually focused on the adsorption process and behaviors, but ignored the conformational changes of the adsorbed protein. Fortunately, some attempts have been reported to simulate the 4


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protein conformation changes upon adsorption. Liao et al investigated the adsorption behaviors and conformation changes of the basic fibroblast growth factor (bFGF) adsorbed on HAP (001) surface by the method of combination of replica-exchange molecular dynamics (REMD) and conventional MD (CMD).35 Cui et al established the most stable adsorption structure of peptide on HAP by RosettaSurface method combining with MD and predicted the relationship between structures and biological activity of HAP-binding peptide.46 Daggett et al employed MD with raising temperature to investigate the protein folding-unfolding pathway.47 Although above attempts expanded our knowledge about the protein conformation changes at certain extent, the computing methods still needed to be improved. MD with raising temperature has a problem of insufficient configuration of sampling. REMD method can achieve enhanced sampling for energetically favorable adsorption states, but it costs huge amount of calculation. On the other hand, RosettaSurface method can obtain representative samples with the lowest global energy and reasonable computational cost. Thus, it would be one of the practical methods for protein conformation computation. In this study, molecular docking and MD simulations were adopted to explore the conformational changes of BMP-2 homodimer on the HAP (100) surface which is one of the most important planes of HAP crystalline,40-41,

48-49

and the adsorption

behaviors of BMP-2 with energy favorable conformations. It would be expected to provide detailed information about the effects of HAP surface structure on the BMP-2 conformation changes and the corresponding adsorption characteristics. The results will be helpful to further reveal the bioactivity nature and the osteoinduction mechanism of Ca-P bioceramics at the atomic/molecular level. As the prospective study of biomaterials genome research, it also provides a new method for designing and screening of tissue inducible biomaterials such as calcium phosphates. 2. Models and methods 2.1 Models BMP-2. The configuration of BMP-2 homodimer was obtained from Protein Data Bank (PDB id 1REW), which contains two active regions, wrist epitope and knuckle epitope.10 The receptors and all crystallographic water molecules were removed with resulted BMP-2 model containing two homo chains, as shown in Figure 1A. 5



HAP (100) Surface. The initial molecule of HAP with space group P63/m was taken from the American Mineralogist Crystal Structure Database. The unit cell parameters are a = b = 9.424 Å and c =6.879 Å.50 The HAP (100) surface51 was prepared as the target model, as shown in Figure 1B. The material size was 18 Å x 130 Å x 130 Å in the x, y and z directions. Total number of atoms was estimated over 30000.

Figure 1. (A) The dimer crystal structure of BMP-2 in Protein Data Bank (PDB id 1REW). (B) HAP (100) surface structure: Side view and top view.

2.2 Methods Docking Method: RosettaSurface was adopted to implement the docking screening. It is a computational tool that simultaneously optimizes protein folding, side chain conformation, and rigid body orientation on solid surfaces. The method is based on the algorithm of all-atom Monte Carlo plus-minimization search and adds a weighted linear combination of van der Waals, electrostatic, hydrogen-bond and implicit solvent interaction on the basis of the all-atom score function of protein folding. It converges to a set of structures with low energy and favorable entropy. At present, this method has been applied in the interaction of peptides and HAP crystal face.52-55 Force Field: The BMH parameters derived from Hauptmann et al56 was usually applied in studying interactions of proteins and HAP surfaces.9, 38, 43-44, 46, 49 However, the BMH parameters have been considered to be particularly appropriate to describe the bulk properties of HAP.43-46 Recently, new parameters, namely INTERFACE force field (or IFF),57 were specifically designed to describe the interface properties of HAP, and thus have been applied to tackle the interactions between HAP surface and biological molecules.57-58 Previously, we even applied this force field to construct an ordered-to-disordered structure for HAP upon stimulated annealing simulation.59 Herein, the IFF parameters,57 which have been integrated in to CHARMM2760-61 6


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force field, were employed in understanding interactions between BMP-2 and HAP (100). In addition, HAP crystals were semi-fixed rather than completely fixed through that no force was applied to the surface of about 12 Å. The intramolecular and intermolecular interactions were described by CHARMM27 all atom force field for BMP-260, 62 and TIP3P water model.63 2.3 Simulation Details The corresponding protein docking and conformational screening were carried out using the RosettaSurface52, 64-65 module implemented in the Rosetta 3.4 software65 with an implicit solvation model. Dictionary of Secondary Structure of Protein (DSSP)66 software was adopted to do the secondary structural analysis. All obtained complex models are further subjected to Cluster Analysis67. In order to have sufficient samples for screening, total of 1000 adsorption models of BMP-2 with different orientations on HAP (100) surface were obtained in this study. As we have mentioned above, the BMP-2 has two epitope sites, knuckle and wrist, with different physiological functions. Therefore, two different models, which are related with close interactions between two epitope sites and material surface, were screened out from these 1000 conformers. Subsequently, classical MD simulations were applied to get the target models fully relaxed. The secondary structure of BMP-2, adsorption characteristics/stability were then analyzed on the basis of the resulted trajectories. All MD simulations were performed by LAMMPS suite of program.68 The initial structures of HAP and BMP-2 were fully solvated in a pre-equilibrated TIP3P water box (100Å x 130Å x 130Å) using the GROMACS 4.6.7 genbox tool.69 Chloride and sodium ions with a concentration of 0.1 M were added to neutralize the whole system. Firstly, the energy minimization was performed by the method of the conjugated gradient.70 Subsequently, as long as 100 ns MD simulations were executed at 310 K with Nosè-Hoover temperature coupling under the NVT ensemble.71-72 The Verlet leapfrog algorithm was applied to deal with the equations of motions at 2 fs time step. The particle-particle-particle-mesh (PPPM) algorithm was adopted to treat the long-rang electrostatic interaction.73 Molecular configurations were visualized by VMD 1.9.1.74 In order to evaluate the binding affinities of BMP-2 molecule by HAP for different conformers, the potentials of mean force (PMFs) for desorption processes were further calculated by SMD.75-77 The BMP-2 molecules were slowly moved to 7



retain the quasi-static states.78-80 Typically, the last frame from the MD trajectory was extracted as the initial structure for SMD simulations. The constant velocity mode of pulling was adopted in current simulation. In the process of SMD, HAP atoms were fixed and a uniform pulling force was acted to atoms on the backbone of BMP-2 to make the BMP-2 move at a constant speed. Water molecules moved freely. The system was calculated under periodic boundary conditions. The spring constant was set at 20 kcal mol-1Å-2, and velocity (ν) was assigned to 10-5Å fs-1. 3. Results and Discussions 3.1 Docking Structure Evaluation RosettaSurface provides a suitable method to investigate and screen the most possible protein adsorption state on the material surface. In this work, total of 1000 docking structures were firstly generated by RosettaSurface module. Subsequently, these structures were divided into three groups according to the different adsorbed active regions of the BMP-2 as shown in Figure 2 and Supporting information Table S1. They were named as Model Ws, Model Ks and Others, respectively. Model Ws

Figure 2. The 2D pie chart of the proportion of various adsorption models in 1000 docking structures.

represented models with the adsorbed residues mainly located in wrist epitope of BMP-2. Similarly, the models with adsorbed residues mainly located in the knuckle epitope were called Model Ks. States not belonging to these two models were grouped into Others. As we have described above, the models related with absorption states of knuckle and wrist epitopes on the HAP surface deserve particular attentions. Therefore, two optimal models (shown in Figure 3A&B) were then screened out from these docked structures with the lowest 8


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Figure 3. The initial models of MD simulation by Rosetta docking approach (A) Model W and (B) Model K. All residues belonging to wrist epitope are colored in yellow, while red color for residues of knuckle epitope are also labeled.

interaction energies and the greatest number of contacting residues belonging to either wrist or knuckle region. The screening results data were shown in Table S1 of Supporting Information. For the screened Model K, BMP-2 mainly contained 39.9% for -strand and -sheet, and 17.9% for the helix. There are 11 residues occurring in the adsorbed amino acids including Ala34, Pro35, Pro36, His39, Phe41, His54, Ile87, Ser88, Leu90, Asn95 and Glu109. These residues mostly belong to the knuckle epitope, namely the region of binding for the type II receptors. Although the secondary structure of BMP-2 in Model W was similar to Model K, the binding residues were found to be Asp25, Val26, Gly27, Trp31, Phe49, Pro50, His54, Val70, Ser72 and Asn95. These are mainly seated in wrist epitope, which can recognize type I receptors. Thus, the conformation and orientation of BMP-2 in the two optimal models were basically in line with the binding mode of the interaction with cells, in which they exerted different biological properties. Of course, only docking simulation cannot tell which one, Model W or Model K, is the closest to the practical adsorption state. Due to that the explicit water molecules and ions cannot calculated in RosettaSurface, it might result in simulations that were relative different from the realistic physiological environment. In order to reproduce the interactions between atoms, MD simulation is necessary to verify the stability and rationality of two docking optimal models.

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3.2 MD Simulation of BMP2 Adsorption 100 ns MD simulations were performed in the explicit water model to observe the adsorption dynamic behaviors and the conformational changes of the two optimal docking structures. Figure 4 showed the changes of the model structures along the

Figure 4. (A) The structure change and active residues for the Model W during the MD simulation of 100 ns. (B) The structure change and active residues for the Model K during the MD simulation of 100 ns. Color designation is same as in Figure 3.

simulation time, and the relative position of BMP-2 active sites was also included. Meanwhile, it was interesting to note that the surface of HAP was partially dissolved by simulating with IFF force field parameters. PO43- and Ca2+ ions on the surface area became gradually disordered as shown in Figure 5A&B. It was found that the material surfaces of both models showed disordered layers with different degrees. The thickness of disordered layer was estimated to be about 15.2 Å for Model W, while about 17.6 Å for Model K, respectively. This result could be consistent with the

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Figure 5. The disordered HAP (100) surfaces of IFF force field displayed using VMD quicksurf module. (A) Model W and (B) Model K. All Ca2+ are colored by cyan, the PO43- ion in red and OH- ions are labeled in pink.

surface characteristics of nanomaterials studied by Jäger81 and Bertinetti82 through UHRTEM. They revealed that solid materials had an amorphous and disordered layer of 1-2 nm thickness on the surface after high temperature sintering treatment. At the same time, this phenomenon was also revealed in the simulation by us.59 To this point, current results might ensure us to apply the IFF force field for further simulation of proteins interacted with the HAP surface. In addition, the Root Mean Square Deviation (RMSD) of BMP-2 backbone atoms revealed that all systems had reached equilibrium finally as shown in Figure 6. For Model K, its RMSD value was about 1 Å with pretty small fluctuation, which indicated that the conformation of BMP-2 was relatively stable. The RMSD of Model

Figure 6. The RMSD of the BMP-2 backbone atoms for Model W and Model K along the MD simulation time.

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W was about 2 Å with significant fluctuation, but still in the equilibrium state. Much larger fluctuation than that of Model K manifests that BMP-2 skeleton movement of Model W should be more intense. Compared with the secondary structure of the initial docked BMP-2, the ratio of each secondary structure of the two models showed distinct fluctuations throughout the MD simulations, as shown in Figure 7. The β structures accounted for the majority

Figure 7. The percentage of second structure of protein during the MD simulation. (A) Model W and (B) Model K.

in the two models. In particular, the percentage of β structures in Model K showed a significant increasing trend (25.7% ~ 34.0%). However, for the structures of helix and turn, the change of its proportion showed a downward trend (13.1% to 9.7%, 10.2% to 3.40%). From the point of view of energy, the instable helix and turn structures was easy to change to a more stable β structures in the simulation process.83-84 It is worth noting that the knuckle epitope is mainly β-sheet structures, while the wrist epitope is mainly helixes including place-helix and pre-helix loop. The proportion of Model K changes in the whole secondary structure is about 4%. The proportion of secondary structure can affect the intermolecular recognition even the stiffness of the biomolecules.83 But at present, there are no more reports and data to interpret the effects of changes in the secondary structure proportion of BMP-2 on its biological activity. Further studies are needed in the future works. At the same time, residues around the adsorption sites of BMP-2 were also changing in accord with evolution time, as shown in Figure 8. Initially, the residues of BMP-2 responsible for adsorption in Model K are located between the β-7 (Ser88, Leu90, Leu92) and β-8 (Lys97, Leu100, Lys101, Asp105, Glu109) regions, which

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belong to knuckle epitopes of BMP-2. On the other hand, the adsorption residues in Model W are mainly located in wrist epitope. With the simulation proceeding, the

Figure 8. The interacting residues of Model W on the HAP(100) surface during the MD simulation. (A) 500 ps, (B) 1 ns and (C) 10 ns. All name of the residues on the wrist epitope are colored by black, the name of residues on the knuckle epitope are colored by blue.

original residues (including Asp25, Gly27, Asp30, Trp31, Pro50, Val70,) responsible for adsorption moved away from the surface gradually. Meanwhile, those residues (including Glu94, Asn95, Glu96) in knuckle epitope area were getting close to the surface and contributed to new recognition modes. At 500ps, residues such as Asp25, Gly27, Asp30, Trp31, Pro50 and Val70 were mainly adsorbed on the crystal surface in the Model W, belonging to the wrist epitope. Then, the number of residues in the wrist epitope gradually decreased at 1 ns. At last, the interacting residues with the surface were located mainly in the knuckle epitopes, which were mainly Glu94, Asn94 and Glu96 at 10 ns, as shown in Figure 8. In fact, according to Figure 9A, we could find that the number of residues around the adsorption sites in wrist epitope

Figure 9. The number of interacting residues on the HAP(100) surface during the MD simulation. (A) Model W and (B) Model K. 13



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decreased continuously before stable adsorption. Overall, the number of the adsorption sites in knuckle epitope was more than that in wrist epitope in most of simulation time. Interestingly, for Model K, we could see from Figure 9B that the recognition of BMP-2 by HAP surface should predominantly attribute to residues belonging to knuckle epitope. To this point, we might simply postulate that the HAP (100) surface might prefer binding knuckle epitope of BMP-2. The residues responsible for recognition in Model K were mainly constituted by Asp, Lys and Glu, which could have strong electrostatic interactions with ions of PO43- and Ca2+ on HAP (100) surface. On the other hand, the interactions between protein and material surface in Model K mainly occured between Ca 2+ ion and residues of Asp and Glu. Indeed, the distances between Ca2+ and the carboxylate group of glutamic acids were calculated to be around 2.6 Å (Table 1) for two models, which Table 1. The distances between surface atoms and adsorbed residues during the MD simulation.

Distance(Å)

Model W

Model K

Glu94-Ca2+

_

2.63±0.11

Glu109-Ca2+

_

3.03±0.86

Asp53-Ca2+

_

3.04±0.81

Lys97-PO43-

_

3.11±0.71

Asp93-Ca2+

_

4.89±0.51

Glu96-Ca2+

2.65±0.14

_

Lys97-PO43-

3.20±0.89

_

clearly indicates strong adsorption states in our simulation. However, according to Table 1 and Figure 10, pretty weak -NH2-PO43- interactions can be found when the adsorption of BMP-2 on HAP (100) surface occurs. Current results are then not inconsistent with previous studies on the interactions between proteins and HAP surfaces.9, 43-44 In addition, due to the different surface properties and protein types, 14


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the optimal adsorption orientation and preferred adsorption sites might be different. For example, Dong et al have found that it was impossible for Glu94 of

Figure 10. Snapshot representations for the complex structures of BMP-2 adsorbed on HAP (100) surface at 100 ns extracted from MD trajectories. Possible interactions between ions on HAP surface and charged residues from BMP-2 are also given for clarification. The main residues interacting with the surface are plotted in ball-and-sticks style. (A) Model W and (B) Model K. Distance units are Å.

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BMP-2 to become a major adsorption residue due to its orientation. However, on the basis of our simulations, Glu94 was the main adsorption site in Model K, while Glu96 was the main adsorption site in Model W. It can be seen that the screening and optimization of the initial orientation and conformation play an important role in the simulation research of the interaction between proteins and material surfaces so that the results would be closer to reality.85-87 3.3 BMP-2 Desorption by Steered MD Simulation Although classical MD simulation could provide some insights into the binding performance of proteins on material surface, it could give contributions of individual residues to the binding affinity. Steered MD (SMD) approach is one of appropriate methods to solve this problem. In fact, the combination of SMD and MD have previously been applied to tackle this issue involved other BMPs on HAP.9, 44 Herein, the SMD was also employed to further reveal the adsorption mechanism of BMPs on HAP based on our newly constructed HAP/BMP-2 complex models. Surprisingly, the monomer model of BMP-2, which bears its biological activity in dimer form10-13, was widely adopted in previous studies. Lack of a complete picture of the protein, deviations should be expected between simulation results and practical experiments. Recently, Huang et al88 systematically studied the adsorption and desorption behaviors of BMP-2 on a nanostructured HAP surface by combining MD simulations and SMD simulations. The results showed that the nanostructured morphology of HAP surface played an important role in the dynamic behavior of BMP-2. However, their simulations provided no further binding information to the BMP-2 functional sites of knuckle and wrist epitopes, since the initial models in their work were prepared via an artificial positioning way. In this work, we will try to understand the behavior of those residues around the major binding epitopes during protein desorption process. On the basis of our MD results presented above, we can easily find out that the Model W is not stable. Number of residues in wrist epitope is even less than those in knuckle epitope during the MD simulation. Therefore, we then simply choose not to further investigate the desorption process of BMP-2 from HAP surface using the Model W as the initial structure. However, relatively stable material-protein complex model can be located in the MD simulation when knuckle epitope directly interacted with HAP (100) surface. Without loss of generality, we simply selected the last frame 16


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structure from the MD trajectory in Model K as the initial model in our SMD simulation. The change of free energy in this process and the adsorption capacity of related sites were analyzed according to the potential of mean force (PMF). The results showed that the orientation of BMP-2 varies significantly in the period of 500 ps to 4000 ps, as shown in Figure 11A. The orientation gradually changed to upright from parallel to the HAP (100) surface at first. During the stage of 300-800 ps, two glutamic acids of Glu109 and Glu83 were desorbed from the surface. Due to the presence of two residues with negatively charged carboxylate group, the measured pulling force was quite large (Figure 11B). At the stage of 1500-2800 ps, the peak of

Figure 11. (A) Potential of mean force (PMF) and orientation evolution of BMP-2 during desorption process from the HAP (100) surface simulated using SMD approach for Model K. (B) Pulling forces with respect to Pulling Constant Velocity time upon SMD simulation for the Model K.

desorption appeared in Fig.11 revealed that Asp53 and Asp93 desorbed. After 3200 ps, the last adsorbed residue was Glu94, making the BMP-2 stick to the surface of HAP (Figure 11A&B). Surprisingly, this interaction was so strong that it could resist the external pulling force of SMD as long as 3ns. After 3 ns, the PMF and pulling force fluctuated around a fixed value, which indicated that BMP-2 was totally desorbed from the HAP surface and no longer had interactions with HAP. The SMD results showed that Glu94 had the strongest adsorption, which was consistent with the results of previous MD simulation86-87,

89.

Meanwhile, our simulation also suggested the

adsorption of BMP-2 on HAP was dominated by the acidic residues containing the 17



carboxylate group. It is worth noting that acidic residues account for about 20% in the knuckle epitope of BMP-2. Reasonably, the knuckle epitope had more stable and stronger adsorption on HAP (100) surface. Moreover, BMP-2 with knuckle epitope adsorbing was more likely to be the favorable orientation. It could allow BMP-2 wrist epitopes to be more exposed to environment, and thus have better chance to bind the type I receptors so that exert the bioactivity of osteoinduction. In summary, results presented above showed that the knuckle epitope region of BMP-2 could form much more stable interactions with the HAP (100) surface more stably, making the wrist epitope upward. This adsorption orientation was more conducive to that the wrist epitope exposed to specifically recognize type I receptors on the stem cell membranes, and exert biological activity (Figure 12). Thus, the biological function could be regulated through the surface modifications of biomaterials.

Figure 12. Graphical representation of the HAP (100) surface effects on the SMAD signaling pathway.

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4. Conclusions Although high-throughput virtual screening method has been widely accepted in drug design,90-92 the applications of such a scenario are still rare46,

52-54

in material

design or investigation of interactions between solid surface and biological systems. In this work, we applied the screening technique, which is implemented in Rosetta software, to prepare the initial models for subsequent MD and SMD simulations. It is believed that such kind of automatic screening method could largely avoid the error brought by manually docking proteins on material surface. The application of RosettaSurface module can not only improve the efficiency and accuracy of materials genes screening, but also promote the development of materials genome on the theory and technology. Meanwhile, the usage of BMP-2 homodimer model and HAP IFF force field could provide more accurately addressing the functional roles of knuckle or wrist epitope with the help of implanted HAP bioceramics. The results showed that there was no obvious change in the conformation of the BMP-2 after docking, but the orientations changed a lot. The MD and SMD results also indicated that the HAP (100) surface prefer to adsorb the knuckle epitope instead of the wrist epitope. To this point, the wrist epitope or the active sites of type I could have much more opportunity to exposure to the environment and easily interact with type I receptors to exhibit its biological activity. All the calculations provided the molecular and atomic information to better understand the mechanism of osteoinduction from the perspective of protein adsorption. Of course, we have to point out only HAP (100) surface and BMP-2 homodimer as the target systems were applied in our work. Limited systems cannot provide complete information to address how HAP affects the absorbates or osteoinductive mechanisms. However, the applications of this high-throughput screening technique in the protein absorption on HAP surface provide a chance to extend to studies on more biomaterial forms and proteins. Furthermore, it might also provide materials genetic screening approach for the design and preparation of novel tissue induction materials.

Notes The authors declare no competing financial interest. 19



Acknowledgments This study was supported by the National Key Research and Development Program (no. 2016YFB0700801). Some of results described in this paper were obtained on the National Supercomputing Center of Guangzhou. Supporting Information The scoring energy of the 10 lowest docking structures. This material is available free of charge via the internet at http://pubs.acs.org/.

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