Nucleation of Biomimetic Hydroxyapatite Nanoparticles on the Surface

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C: Physical Processes in Nanomaterials and Nanostructures

Nucleation of Biomimetic Hydroxyapatite Nanoparticles on the Surface of I-type Collagen: Molecular Dynamics Investigations Zhi-yu Xue, Mingli Yang, and Dingguo Xu J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b10342 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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The Journal of Physical Chemistry

Nucleation of Biomimetic Hydroxyapatite Nanoparticles on the Surface of I-type Collagen: Molecular Dynamics Investigations Zhiyu Xue1, Mingli Yang2,3 and Dingguo Xu1,2* 1MOE

Key Laboratory of Green Chemistry and Technology, College of Chemistry,

Sichuan University, Chengdu, Sichuan, P. R. China 2Genome

Research Center of Biomaterial, Sichuan University, Chengdu, Sichuan

610064, P. R. China 3Institute

of Atomic and Molecular Physics, MOE Key Laboratory of High Energy

Density Physics and Technology, Sichuan University, Chengdu, Sichuan, 610065, P. R. China *

To

whom

correspondence

should

be

addressed:

Tel:86-28-85406156

1

ACS Paragon Plus Environment

[email protected]

The Journal of Physical Chemistry 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

Abstract Biomineralization is one of the most widespread phenomena in biological processes, which can facilitate to harden or stiffen existing tissues in living organism via forming minerals. Understanding the mechanism of biologically controlled or induced mineralization would be particularly useful in the development or optimization of biomaterials. The nucleation of minerals stays at the core position during mineralization. In this work, the molecular dynamics simulation approach was employed to tackle the nucleation process of hydroxyapatite in aqueous phase with the type-I collagen as the seed. In our simulation, we can offer strong evidence to confirm the formation of Ca-P clusters on the collagen surface, and predominantly around the charged residues, especially when the Glu-Arg pair occurring at the same region. Before the nucleation on the collagen surface, small Ca-P clusters could be generated initially in solution with characteristics of Ponser's Cluster. Moreover, the surface free energy calculations could confirm that the energy would continuously decrease with the increasing of cluster size. Our simulation also indicates that different collagen properties could lead to different morphology of final amorphous calcium phosphate clusters.

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1. Introduction Biomineralization is one of the most widespread phenomena in biological processes, which can facilitate to harden or stiffen existing tissues in living organism via forming minerals1-6. Generally, more than 60 different minerals have been identified in organisms. Among them, minerals based on phosphate and carbonate salts of calcium are widely distributed in vertebrates, which are further in conjunction with some organic polymers like collagen and chitin to support bone structures. In fact, the bone tissue engineering involves intensive mineral growth during the bone repairing or regeneration7. Therefore, understanding mechanism of biologically controlled or induced mineralization would be particularly useful in the development or optimization of biomaterials. The nucleation is often thought to be the key step in biomineralization1,

8-10.

Currently, two fundamental nucleation theories in solution have been proposed, namely non-classical and classical nucleation theories.11-13 Quite interestingly, the recent in situ transmission electron microscopy (TEM) imaging14 of the calcium carbonate nucleation revealed that two possible nucleation pathways might exist together. The nucleation process or regulation of morphology of minerals with the help of versatile biological systems seems to be more complicated. It has been suggested that the nucleation and crystallization of hydroxyapatite in physiological environment can be initiated or controlled by proteins like collagens.15, 16 In particular, for the growth of calcium phosphate or hydroxyapatite (HAP) in bone or enamel, several proteins have been found to be important, e.g., type I collagen17, bone saloprotein18, osteopontin,19,

20

and ameloglenin.21 Therefore,

understanding the mechanism of nucleation of calcium phosphate on protein surface is not just of theoretical importance, but also has possible applications in bone repair 3



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engineering and the treatment of pathological mineral formation in biological environments. In this work, we will focus on the functional role of human type-I collagen protein in the nucleation of HA. Basically, the collagen is one of the major organic components of the bone extracellular matrix (ECM).22,

23

Their important

roles in bone repairing24-28 and osteoinduction29 have long been recognized. Among collagens, type-I collagen has the highest content in the human body,17, 30 which is also one of excellent biomaterials due to its low immunity and good biocompatibility. The type-I collagen is a 300 nm heterotrimer triple-helical molecule consisting of two α1 chains and one α2 chain.17, 31-33 There is another homotrimeric isoform of type I collagen, which consists of three same α1 chains.34, 35 Numerous experimental studies6, 9, 10, 15, 16, 24, 36, 37 and theoretical simulations38-44 have been devoted to understand HAP mineralization mechanisms induced or controlled by collagen proteins. Traditionally, the amorphous calcium phosphate (ACP), has been widely accepted as the precursor phase for final crystallization of hydroxyapatite.45 Moreover, with the help of biological additives,46-49 citrate50, 51 and supramoleclar template,52 the enamel- or bone-like apatites could be produced by nanodimensional hydroxyapatite and ACP. A recent cryogenic transmission electron microscopy (cryoTEM) study53 on the biomimetic surface induced calcium phosphate crystallization clearly confirms the existence of the ACP precursor phase. On the other hand, the functional roles of collagen-I in the nucleation, growth, formation of bone apatite have been studied to some extent.9, 15, 16 In particular, the nucleation sites on the collagen-I seems to be charged residues since they can interact with either Ca2+ or PO43- directly.9 Theoretically, the nucleation process of calcium phosphate regulated by collagen or model peptide has been studied via molecular dynamics (MD) simulations by several groups.38-40,

44

In particular, models using the collagen 4


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mimetic peptide (CMP), which just contains the simple repeat collagen sequence (Gly-Pro-Hyp)n, were applied to mimic the nucleation process in the presence of collagen38, 44. The abbreviation of Hyp means hydroxyproline residue. Of course, the amino acid sequences for those CMP models are much different from human type-I collagen, which is thought to be dominant in bone regeneration or repair engineering7. Therefore, simulations based on CMP models might not provide complete information of the nucleation occurring on the surface of the human type-I collagen. In addition, the type-I “super-twisted” collagen model, which is one of the collagen microfibers structure formed by an electronic

interactions of each single collagen

molecule, was employed by Zhou et al.43 However, some details of the nucleation have not be well addressed, e.g., nucleation sites, the morphology of clusters and the roles of specific charged residues. In this work, we will try to understand the nucleation mechanism of the phosphate salt of calcium with the heterotrimer human type-I collagen participating as the seed. In particular, extensive MD simulations will be applied to understand the formation and growth of ACP clusters. 2. Computational Details 2.1. Collagen Model In general, the type I collagen protein consists of three α-chains and each chains have around 300 Gly-Xaa-Yaa triplets.31 Gly is a fixed amino acid in the smallest unit and some charged residues but not proline or hydroxyproline could occupy the Xaa and Yaa positions. In vertebrate extracellular matrices, the X-ray structure (PDB code 3HR2) with the full length of collagen type I protein was reported.17 However, this structure only contains backbone α-carbon atoms. In order to construct the initial triple helix collagen model, the THeBuScr script54 was applied on the basis of the 5



primary sequence of human type-I collagen (PubMed entry number NP_000079 for α1 chain and NP_000080 for α2 chain). Considering the length of the whole collagen protein, expensive computational cost might restrict it to be treated using all atom molecular dynamics simulation. We then divided the whole sequences into thirteen even short segments. To avoid the possible boundary effects, followed by the strategy proposed by Chang et al.,34 the same six residues of GFPGPK were added to N termini and other same six residues of GEQGPA were added to cap the C termini of three chains, respectively. Once the triple helix structure was obtained, the side chains of the collagen model was complemented using Modeller (version 9.17).55 Subsequently, all thirteen short collagen models were fully relaxed by geometry optimization and molecular dynamics simulation (40 ns) using GROMACS version 4.6.7 package.56 Each model collagen was fully solvated in the pre-equilibrium rectangular TIP3P57 water box with the size of 290 Å × 80 Å × 90 Å. The non-bond interactions cutoff was set to be 12 Å. Sodium and chloride ions were added to neutralize the target system. The integration step size was set to be 2 fs, pressure at 1atm and temperature at 310K. The particle mesh Ewald (PME) algorithm58 was applied to treat the long range electrostatic interactions. In all calculations, the CHARMM2759 force fields were employed to describe the whole collagen protein. It should be pointed out that the collagen protein features a non-standard residue of hydroxyproline (Hyp). We then used the modified CHARMM27 fore fields which include the charges60, bond, angle and dihedral parameters of Hyp based on the standard AMBER general atom fore filed (GAFF).38 2.2. Nucleation MD In this work, we will try to understand the nucleation of the salt of calcium phosphate on the surface of collagen protein. Therefore, ions of calcium and 6


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phosphate should be added into the solution properly. The normal concentration of calcium ions is 2.5-2.75 mM in our body fluid.40,

61

However, much higher

concentration (0.7 M) was employed by several groups to enhance sampling and accelerate the simulation.38-40,

62

One of example structures for collagen models

complexed with ions was displayed in Figure 1. In our simulation, the 0.7 M calcium ions were randomly added into the system, and the concentration of other ions like phosphate and hydroxyl ions followed the stoichiometry of the HAP crystal. 0.1M sodium chloride was added to neutralize the system, which mimics the human body physiological environment. The size of unit periodic box is around 290 Å × 80 Å × 90 Å for each short collagen model. This could result in a total atom number of the whole simulated systems is over 2 million. After we obtained the collagen models mixed with calcium and phosphate ions, the LAMMPS suite of program63 was employed to understand the nucleation process regulated by collagen protein. Molecular configurations were visualized using VMD 1.9.164. First of all, total of 10000 step conjugate gradient minimization was applied for geometry relaxation. MD simulations were carried out in the isothermal-isobaric ensembles (NPT) firstly (0.1 nanosecond) and followed by the 10 nanoseconds canonical (NVT) ensemble simulations using Nose-Hoover thermostats. The long-range electrostatic interactions were treated using the PME algorithm65 with precision of 10-4.

The temperature is set to be 310 K and the pressure is 1 atm,

respectively. SHAKE algorithm66,

67

was used to maintain all covalent bonds

involving hydrogen atoms of water molecules. Newton’s equations of atomic motion were integrated with 2 fs time step by the Verlet algorithm. In this work, the recent developed INTERFACE force field, namely IFF,68 was used to describe motions of calcium, phosphate and hydroxyl ions. 7



3. Results 3.1 Precipitation of Clusters First of all, due to expensive computational cost of all atom MD simulation on the full size I-type collagen, we have simply divided it into 13 segments. To avoid the artificial effects, we then added the same terminal peptides to the terminus for each segment. During the initial stage of MD simulations of the collagen models, all 13 models can maintain their triple helix structure quite well. All nucleation dynamics will then carried out based on these models. Ten nanoseconds MD simulation is sufficient to observe the precipitation of the cluster constituting by calcium and phosphate ions. From Figure S2, after ten nanoseconds MD simulation, all 13 segments of collagen models can still keep their stable triple helix structures. Impressively, clusters containing ions of Ca2+, PO43- and OH- with different size accumulate and spread around the surface of each collagen segment. Since the ions in solution is in supersaturated status, the ions aggregated both on the collagen surface and in solution quickly. Notably, the size of clusters on the collagen surface is significantly larger than those in solution. Figure 2A depicts the average size comparison of the clusters in solution and on the collagen surface. Clearly, the precipitation or the size of calcium phosphate in solvent will reach nearly equilibrated after a quite short simulation time, whereas the clusters on the surface can continuously aggregate along the time course. Therefore, we might simply anticipate that the phosphate salt of calcium could finally precipitate, although we have no idea how long it will take. The collagen in solution can facilitate cluster aggregation and even biomineralization for calcium phosphate without question. On the other hand, if we carefully check the components of the cluster on the collagen surface, we can find 8


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that the sodium and chloride ions never appear in the clusters. This also means the cluster has the formula of Cax(PO43-)y(OH-)z. Another interesting phenomenon could be observed from Figure S2 is an uneven distribution of Ca-P clusters on the collagen surface. Figure 2B plots the occurrence rate of ions aggregation on the collagen surface, which is defined as the ration of number of ions occurring in clusters and number of total ions in solution for each segment. The possibility of formation of the cluster in the 7th segment collagen model seems to be the largest. On the other hand, those clusters with different sizes are only located at some specific binding sites, which indicate the selectivity of nucleation site. More details will be discussed in below. 3.2 Properties of Clusters As we know, the calcium and phosphate ions will generate clusters in solution when approaching a level of supersaturation. Indeed, such phenomena have been observed for calcium carbonate mineralization in liquid phase.69, theoretical computation69 and experimental observations70,

71,

69

On the basis of

the onset of CaCO3

mineralization shows a clear non-classical nucleation process with distinct sign of amorphous calcium carbonate (ACC), which contradicts classical nucleation theory with unstable clusters. On the other hand, recent experiments also show that the ions can aggregate to generate ACP with the different morphology during the mineralization of hydroxyapatite assisted by collagen proteins.15, 16, 36 In this work, we have systematically investigated the nucleation process in the liquid phase with the presence of collagen protein. In order to understand the properties of the generated clusters along the MD simulations, we have to carefully investigate their size, purity and morphology, since larger and denser clusters should be the key step of nucleation and final crystallization.11 9



The size of these clusters on the collagen surface area typically ranges from 100 to 835 atoms within 10 ns MD simulation. Meanwhile, their topologies are dramatically different. Figure 3A plots one example of Ca-P clusters generated on the collagen surface. Interestingly, this cluster even features a distinct porous structure. If we recall that the real bone is a porous-like material constituted by calcium and phosphate ions, current topology might be a spontaneous result regulated by proteins like collagen. On the other hand, we randomly selected three clusters on the collagen surface from the 2nd, 4th and 7th collagen segments to understand the evolution of Ca/P ratio along the simulation time. As shown in Figure 4A, although in the initial nucleation stage, the Ca/P ratio differs dramatically among three clusters, the ratio finally reaches nearly the same value around 1.3 ~ 1.5. Recently, Jahromi et al48 conducted one experiment to investigate the effects of charged amino acids in the crystallization of hydroxyapatite, in which the Ca/P ratio was found to be 1.35 after three days-aged precipitation in solution with the presence of either Glu or Arg. Impressively, the statistically averaged Ca/P ratio for all clusters on the collagen surface is calculated to be about 1.37 in our simulation, which agrees with the experimental data very well.48,

72

At the same time, we also summarized the size

evolution of these three clusters using the number of atoms along the simulation time in Figure 4B. As we can see, they all fall in the range of the critical nucleus size, which should contain about 100 to 1000 atoms in the cluster.11 On the other hand, the Ponser’s cluster73, which has the chemical formula of Ca9(PO4)6, was usually considered to be the fundamental unit of the ACP, and thus critical for the nucleation of apatite in solution. If we carefully check the stoichiometry for all of Ca-P clusters formed on the collagen surface, we can find they all can be expressed as the form of multiple Ponser’s clusters. For example, two clusters shown in Figure 3A and S3A, 10


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respectively. Both can be grouped into Ponser's clusters, i.e., [Ponser]12 for Ca108(PO4)72(OH)10 from the seventh segment, while [Ponser]20~[Ponser]21 for Ca182(PO4)127(OH)9 from the second segment if we only consider calcium and phosphate ions. To this point, we could believe all clusters formed in this work should be considered to be the experimentally proposed ACP.45 Another interesting issue is the distribution of water molecule around the clusters.45 We can also notice some water molecules distribute inside the pore. One example was depicted in Figure 3B for all water molecules within the distance of 5 Å around the cluster. Figure S4 gives the mean square deviation (MSD) for three types of ions, OH-, Ca2+ and PO43-. Clearly, the movement speed in solution has the sequence of OH- >> Ca2+ > PO43-. This is quite reasonable considering the mass of the ions. Meanwhile, this can also provide a reasonable explanation that during the precipitation, hydroxyl ions are very difficult to be trapped by collagen surface residues, while phosphate groups are pretty easy to be absorbed to collagen surface charged residues. Moreover, once the calcium or phosphate ions are bound to the collagen surface residues, the interaction is quite stable no matter the size of the Ca-P clusters. Typically, the closest distance between Ca2+ and the carboxylate group of Asp or Glu has the range of [2.5, 3.0] Å, while [3.5, 4.0] Å between phosphate group and positive charged group of Arg or Lys. Such distances clearly indicate that the interaction between Ca-P clusters and collagen protein is pretty strong and stable. Further, this also means that the formed Ca-P clusters will stay on the collagen protein surface, but not move to the solvent. Another property to characterize the cluster is the pair distribution function (PDF) for the Ca-P, Ca-Ca and P-P, which could tell us the micro structure of the generated clusters. For comparison, we also plotted the PDFs for both TCP and HAP in Figure 5. 11



Obviously, the PDF for the clusters is disordered no matter at the short or long range zone. However, for the PDF of Ca-P, two clear peaks occur at 3.16 and 3.78 Å, which are pretty close to crystal properties of HAP. Thus, current simulation can show that the generated clusters have the tendency to further grow up to form an ordered crystal structure. Meanwhile, to further characterize the clusters, we then calculated the average distances between atoms in the clusters, which are summarized in Table 1. According to Table 1, the fundamental geometric parameters of the clusters are quite close to the HAP crystal structure. 3.3 Nucleation Process Analysis One of important issues has to be clearly addressed is that the nucleation process for the calcium phosphate assisted by collagen protein. Traditionally, the nucleation mechanism has two proposals in aqueous phase, namely classical and a two-step nucleation pathway. For the first pathway, the ions could directly aggregate together, and finally precipitate in solution. The second pathway indicates that ions could self-assemble in solution to form many metastable amorphous clusters or spherical nano particles, which further aggregate together to fulfil final precipitation11. Recent experimental investigations15,

16, 36

of biomineralization of calcium phosphate

regulated by collagen protein support that the nucleation process has the characteristics of the non-classical nucleation mechanism or two-step nucleation mechanism evidenced by the observation of ACP. In our simulation, we can also have the similar phenomena with experimental observances. To elucidate the performance of ions of calcium and phosphate in the presence of collagen, we then selected one segment model (the 7th segment) from our 12


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simulation for further investigation. In Figure 6, we plotted the evolution along the simulation time course for all calcium and phosphate ions that belonging to the clusters formed on the collagen surface, which is extracted from the seventh collagen segment. Four snapshots at 0, 2, 4, and 10 ns were included, respectively. For clarity, those phosphate ions that finally aggregate together were plotted using the pyramid style, while calcium ions were plotted in ball style. Along the simulation, we can easily identify some calcium and phosphate ions are absorbed by several charged residues. Meanwhile, even in a relatively short time (2 ns), some ions could also aggregate together in solution with a self-assembly way. Quite impressively, nearly 50% clusters could be grouped into Clusters with different size were generated according to Figure 6B. We then statistically summarized the size and stoichiometry form for these clusters in Table S1, in which the Ponser’s cluster was identified according to Ca/P ratio ranging from 1.40 to 1.55. This clearly means the existence of ACP during the collagen regulated nucleation and even final biomineralization. Cluster surface energy is another sign to estimate the stability of generated ΔE

clusters. We calculated the surface energies (),which is defined as 𝛾 = A ,74 for those s clusters bearing characteristics of Ponser's cluster. The model clusters were extracted from the MD trajectory at 2 ns during the nucleation process for the 7th collagen segment. The E can be defined as the energy of a single cluster. The surface area (As) of the cluster was estimated using solvent accessible surface area (SASA) method75. Figure 7 depicts the surface energy. Clearly, the surface free energy shows an opposite tendency with cluster size. For comparison, the first five small clusters, 13



which do not belong to Ponser's cluster, were also included. As we can see, with the size increasing, the surface energies reduced accordingly. Such energetic profile also suggests that the formation and growth of the Ca-P clusters should be a thermodynamic favorable process. If we carefully check all thirteen MD trajectories, we could identify at least three ways for the aggregation and growth of ions to produce clusters. First, some calcium and phosphate ions could assemble around the charged residues to form a small cluster, which could further absorb other ions in solution and become bigger gradually. The second pathway is the ionic clusters will firstly form in solution, which could be trapped by other small clusters formed on the collagen surface. The third pathway is the clusters formed on the collagen surface capture other smaller clusters from nearby sites. The simultaneous existence of these three ways to form larger clusters means the nucleation assisted by collagen protein is complicated and also a competitive ionic adsorption process. 3.4 Analysis of Nucleation Sites As shown in Figure S2, we have noticed that uneven distribution of cluster formation or nucleation of calcium phosphate. More importantly, those sites for the nucleation seem to only occur around those charged residues like Asp, Glu, Lys and Arg. First of all, we counted the participation number of amino acids in the formation of Ca-P clusters during the MD simulation. To avoid artificial estimation, we then included all residues from all thirteen segments within 5 Å away from the Ca-P clusters in our analysis. Such results can be seen in Figure S5, in which we calculated possibilities for all types of amino acids and charged residues of all and separate 14


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collagen segments. Notably, from Figure 8A, the possibilities evolution trends of the ions occurring close to both all residues and only charged residues are nearly the same. Meanwhile, after relatively short MD simulation, the occurrence possibilities around the Ca-P clusters can reach equilibrium. All these results strongly suggest that those charged residues could be the main factors to facilitate the formation and growth of Ca-P clusters. To further illuminate this result, we then summarized all nucleation sites using amino acid sequence analysis in Figure 9. In particular, the red colour represents negative charged amino acids, while the blue colour denotes positive charged amino acids. Since some of the proline residues in collagen protein are replaced with Hyp, we simply labelled them with an abbreviation "P", but coloured by green to distinguish from normal prolines (colored by black). We can see some basic structural characteristics for the type-I collagen molecule. For example, there are same two or three adjacent amino acids of the same charge (Asp/Glu or Arg/Lys) occurring simultaneously in each helix. At the same time, residues with the same charge (Asp/Glu or Arg/Lys) are often situated nearly at the same position of among different helices. Another structural property for type I collagen molecule is that one charged residue and its opposite charged residue often occur in close proximity.10 Such kind of structure could largely affect the ions absorption and further aggregation without question. As we have discussed above, during the precipitation of calcium phosphate, the protein does show its critical role on the basis of the comparison of cluster size in solution and on the collagen surface. According to Figure 9, we can easily find residues in the square circled regions are mainly charged residues (Arg, Lys, Glu and Asp), around which most of the Ca-P clusters were produced. This suggests that 15



residues with both negative and positive charged residues could largely promote the aggregation of ions around themselves. Another interesting phenomenon is nearly all the squared regions contain not only one type of charged residues. To illustrate this, we then plotted the occurrence frequency of Ca-P clusters in Figure 8B upon the group of two residues containing opposite charge, in which the possibility of the nions

accumulation of the clusters can be defined to be ( ntotal ). 𝑛ions denotes the number of Ca2+, PO43- and OH- in the Ca-P cluster around the specific amino acids pairs (Glu-Lys, Glu-Arg, Asp-Lys and Asp-Arg) and 𝑛ions represents the total number of Ca2+, PO43- and OH- in solution. For comparison, we also included two more pairs with same charge like Glu-Asp and Arg-Lys. Impressively, the occurrence rate of Glu-Arg pair seems to be dominant compared to all other cases. On the other hand, we could deduce that the Glu-Arg pair should be the major factor to facilitate the formation of Ca-P clusters. Our results can also show that the nucleation process controlled by type-I collagen molecule has certain directionality. Interestingly, recent studies by Jahromi et al48, 49, 76 strongly show that individual dissolved amino acids could inhibit the precipitation of HA via chelating interactions with either Ca2+ or PO43-. However, the combination of Glu and Arg show less effect in controlling HA nucleation but inhibit the HA crystallization. Clearly, in order to completely understand the functional roles of the biological additives in orientating the HA nucleation, a proper physiological environment might be highly necessary. The detailed interaction patterns between those Ca-P clusters and collagen surface charged residues deserve special investigations. Without loss of generality, we choose two Ca-P clusters formed on the collagen surface of the second and seventh collagen segments for further investigations, which have the stoichiometry of 16


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Ca108(PO4)72(OH)10 and Ca182(PO4)127(OH)9, respectively. The detailed interactions between two clusters and nearby residues were depicted in Figure 10 and S6. The calcium ions can interact with the carboxylate group of negatively charged amino acids with the non-bond distance around 2.7 Å, whereas the distances between the phosphate ions and positively charged guanidine group of Arg or amine group of Lys are typically less than 4.0 Å. Such short distances could strongly suggest strong electrostatic interactions between Ca-P clusters and collagen surface. In fact, the electrostatic interactions have been proposed to be the major driving force for the interactions between protein and Ca-P based biomaterials.77-79 On the other hand, we can also identify the calcium ions will always interact with only one oxygen atom of the carboxylate group. Similarly, only one of oxygen atoms of phosphate group will be in direct contact with Arg or Lys residues. Moreover, the average coordination number of P around Ca is calculated to be 5.48 for the cluster in Figure 10, while 5.22 for the cluster in Figure S6. Both are pretty close to 5.40 in the HAP crystal structure. This may indicate that the clusters are getting closer to the crystal structure when the type-I collagen serves as the seed for the nucleation of calcium phosphate. 4. Discussion In this work, we have systematically investigated the nucleation process of ions of Ca2+, PO43- and OH- with the help of human type-I collagen molecule. The original human type-I collagen molecule has the repetitive unit of Gly-Xaa-Yaa, in which X and Y replaced with some charged residues and hydroxyproline ocassionally. During the MD simulation, we can specifically observed Ca-P clusters with different size and shape formed on the collagen surface. The distance between the cluster and collagen

17



ranges from 2.5 to 4.0 Å, which indicates strong interactions between collagen and clusters. Furhter, during the MD simulation, we can observe some small clusters formed in solution before they migrate to the collagen surface. The stoichiometry of these clusters could have the characteristics of Ponser’s cluster, which is considered to be the prerequisite fundamental unit of crystallization of hydroxyapatite. Therefore, our simulation can offer some solid evidences to support the formation of Ponser’s cluster during the crystallization of hydroxyapatite, although the time scale of our computation cannot reach the limit of crystallization. Another important issue is that the functional role of charged residues in the process of formation and growth of the Ca-P clusters. The nucleation sites with large size Ca-P clusters are mainly around the charged residues, especially the site containing Glu-Arg pair. In other words, the Glu-Arg pair should be considered to be the main factor to promote the growth of Ca-P clusters, and thus be critical in the crystallization of hydroxyapatite. Previously, the mechanisms of nucleation of HAP have been explored to some extents. However, we have to point out that the collagen models used in some previous work are nearly based on collagen mimetic peptide (CMP) model, which contains the repetitive units of Gly-Pro-Hyp. This collagen model can perfectly mimic the tropocollagen style for collagen molecule. However, it shows intrinsically difference from human type-I collagen molecule, e.g., existence of charged residues. Therefore, nucleation simulation of HAP based on this kind of collagen model might not directly provide insights into the functional role of charged residues in the 18


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formation of nucleus or clusters of HAP. To compare the performance of ions of calcium and phosphates in the presence of CMP and type-I collagen model, we further carried out the nucleation simulation when CMP system in solution. The coordinate of CMP can be adopted from the protein data bank (PDB entry code 1CGD). In this work, 26 repeat units of Gly-Pro-Hyp were employed as the collagen model system. All of other MD setup protocols are the same as we did for type-I collagen. The size for the system is 290 Å × 80 Å × 90 Å. Total of 10 ns MD simulation was performed. Notably, we cannot observe any clusters formed on the CMP based collagen surface. As shown in Figure 11, although some clusters seem to be generated in solution, the shortest distance from the protein to the cluster is even longer than 8 Å, which means no direction contact between Ca-P clusters and protein. Since the CMP collagen model contains no charged amino acids, it cannot function as the seed during nucleation of hydroxyapatite. In other word, we can confirm the importance of charged residues once again. The collagen model employed in this work was simply divided into 13 segments. Such kind of treatment can save a lot of computational cost and allow all atom simulation to be feasible. However, our treatment of the collagen moleucle might not provide a complete picture for the HAP nucleation. It would be better to conduct this type of simulation for collagen molecule with full length. On the other hand, it has been well known that the hyman type-I collagen molecule is usually in a microfibril form in real case, and further forms a hierarchical strucutre via some organic crosslinks.31, 80 Of course, the size of such system is much over the limit of all-atom 19



MD simulation could reach. Coarse grained modeling approach might be more approapriate for such large-scale systems. 5. Conclusions Biologically induced or controlled biomineralization represents one of widespread phenomena in biological systems. The mineralization of the salt containing ions of calcium and phosphate has been considered to be particular useful in the bone repairing or the treatment of pathological mineral formation in biological environments. Moreover, the nucleation process of salt of calcium and phosphate is the key step before mineralization. To understand the Ca-P clusters growth process in solution phase in the presence of the human type-I collagen molecule, extensive MD simulation were carried out in this work. In our simulation, we can find that the clusters containing calcium and phosphate ions aggreegate in solution phase and on the collagen surfaces, but with much different size and morphorlogy. Much larger clusters can be found occuring assisted by charged amino acids. Of course, unevenly distributions of Ca-P clusters also indicate that the aggregation of ions containing calcium and phosphate could be induced or controlled by protein surface properties. In particular, the size of the clusters around the pair of Glu-Arg is much larger than other position, which means that the growth of cluster or nucleation should have site specificity or directionality. During the formation of ACP on the collagen surface, we could identify some smaller clusters, which also have the chemical formula of Ponser’s cluster. That strongly supports a non-classical nucleation pathway for the HA nucleation with the collagen-I 20


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as the seed. On the other hand, those clusters aggregated on the collagen surface shows dramtically different morphorlogies. Even we could observe the clusters at some collagen segments have porus structure, which also suggest that type-I collagen does have possibility to induce or orientate the growth of calcium phosphate clusters, although direct connection between the morphorlogy and protein cannot be established so far. We still believe that this could provide a chance to pursue a way to regulate or optimize the mineralization via protein engineering technologies.

Acknowledgement This work was supported by National Key Research and Development Program (No. 2016YFB0700801). Some of results described in this paper were obtained on the National Supercomputing Center of Guangzhou and Supercomputing Center of Sichuan University.

Supporting Information The backbone RMSDs of thirteen collagen proteins along the dynamics simulation time. The generated clusters on the collagen surface for all thirteen segments after 10 ns MD. The details of evolution of cluster (Ca108(PO4)72(OH)10) in the 7th collagen segment. Mean square displacement (MSD) values for ions of Ca2+, PO43- and OHalong the simulation time. The probability of the nucleation on the collagen protein surface during the 10 ns simulation. The summary of Ponser’s clusters occurred at 2

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ns in the seventh segment collagen model. This material is available free of charge via the internet at http://pubs.acs.org.

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Figure 1. An example initial state of collagen model in aqueous phase in presence of

calcium, phosphate and hydroxyl ions.

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Figure 2. (A)The size comparison of two largest clusters in solution and on the collagen surface in the 7th collagen segment model, respectively. For convenience, we only count the number of calcium and phosphate ions occurring in the clusters. (B) The proportion of the occurrences of Ca-P clusters on the collagen surface.

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Figure 3. (A) One typical cluster (Ca108(PO4)72(OH)10) formed on the collagen surface extracted from the last frame of MD trajectories. (B) The water distribution around the cluster with distance of 5 Å.

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Figure 4. (A) Ca/P ratio for selected clusters as a function of the simulation time. (B) Numbers of atoms for these selected clusters.

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Figure 5. Pair distribution functions for the Ca-P, Ca-Ca and P-P of (A) HAP (B) -TCP (C) Cluster in the seventh collagen segment model.

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Figure 6. The evolution along the dynamics time course (0, 2, 4 and 10 ns) for those calcium and phosphate ions that belonging to the clusters formed on the collagen surface, which is extracted from the seventh collagen segment. For clarity, those phosphate ions that finally aggregate together were plotted using the pyramid style, while calcium ions were plotted using ball style.

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Figure 7. The surface energy of clusters in the 7th collagen segment after 2ns MD simulation.

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Figure 8. (A) The probability of the nucleation on the collagen protein surface during the 10 ns simulation. (B) The comparison of occurrence ratio of clusters upon the charge residues pair during the 10 ns simulation.

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Figure 9. Sequence summarization for the nucleation sites. The N-terminal was capped by GFPGPK, whereas the C-terminal part was capped by GEQGPA for three chains in all thirteen collagen segment models. All negatively charged residues (Asp and Glu) were colored in red, while positively charged residues were in blue color. The red square denotes the number of calcium, phosphate and hydroxyl ions in the cluster is over 300 , pink is between 200 and 300, purple square contains 100-200 ions and less than 100 ions for black square region.

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Figure 10.

The details of nucleation site between the collagen protein and and one of clusters extracted from the second segment of collagen models, in which the cluster has the stoichiometry of Ca108(PO4)72(OH)10.

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Figure 11. Snapshot for the CMP model after 10 ns nucleation MD simulation, (A) side view (B) zoom-in view from C-terminus.

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Table1. Average particle-particle distances for clusters on the surface and for HAP crystal. Distances (Å)

Crystal Structure

Clusters

Ca-Ca

3.94

4.48 ± 0.39

Ca-P

3.08~3.67

3.68 ± 0.45

Ca-O(H)

2.38

3.19 ± 0.40

Ca-O(P)

2.34~2.81

3.08 ± 0.35

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TOC Graphic

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Nucleation of Biomimetic Hydroxyapatite Nanoparticles on the Surface

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