Free Energy Calculations of Amino Acid Side Chain Analogues

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Essence of Small Molecule-Mediated Control of Hydroxyapatite Growth: Free Energy Calculations of Amino Acid Side Chain Analogs Zhijun Xu, Qichao Wei, Weilong Zhao, Qiang Cui, and Nita Sahai J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b12142 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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The Journal of Physical Chemistry

Essence of Small Molecule-Mediated Control of Hydroxyapatite Growth: Free Energy Calculations of amino acid side chain analogs

Zhijun Xu*12, Qichao Wei1, Weilong Zhao2, Qiang Cui3, Nita Sahai*2,4

1

College of Chemical Engineering, State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 210009, China.

2

Department of Polymer Science, University of Akron, Akron, Ohio 44325-3909, United States. 3

Department of Chemistry and Theoretical Chemistry Institute, University of Wisconsin-Madison, Madison, Wisconsin 53706-1322, United States. 4

Department of Geology and Integrated Bioscience Program, University of Akron, Akron, Ohio, 44325-3909, United States.

Corresponding author: [email protected]; [email protected]

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Abstract Much effort has been exerted to unravel bone biomineralization mechanism by which the growth of plate-shaped crystals of hydroxyapatite (HAP) is regulated within a fibrillar collagen matrix. Acidic noncollagenous acidic proteins (NCPs) bearing a large number of carboxylate and phosphorylate amino acid residues are effective regulators of HAP crystal morphology. To reveal the energetic and structural essence of the growth-regulation mechanism, we performed here advanced molecular dynamics simulations to obtain adsorption free energies of side chain analogs (SCAs) of acidic and uncharged polar amino acids as well as the corresponding zwitterionic backbone (BB) fragment, and compared the results to those for the entire amino acid molecule. We observed that negatively-charged SCAs (phosphoserine in two protonation states, aspartate and glutamate) and the zwitterionic BB preferentially bind to the HAP (100) surface compared to the (001) face, consistent with [100] being the preferred growth direction for plateshaped HAP crystals. Charged SCAs and zwitterionic BB bind via the formation of salt bridges between the -COO- group and Ca2+ ions on the surface, or hydrogen bonds between the -NH3+ and surface PO43- ions, and adsorption of uncharged, polar serine SCA was thermodynamics unfavorable. Intriguingly, however, binding free energies depend on the number of charged groups rather than simply on net charge. Thus, zwitterionic (net neutral) BB adsorbed more strongly than monovalent Asp and Glu or uncharged Ser SCAs. Secondly, it was more difficult for larger molecules to adsorb presumably because of less favorable enthalpic and entropic contributions required to penetrate the tightly bound surface water layer. Accordingly, Asp adsorbed more strongly than homoionic Glu; and the sum of the SCA and BB binding affinities was greater than that of the entire amino acid molecule. The present results shed light on some previously unidentified subtle effects that the number of charged motifs rather than net charge 2


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and molecule size control a delicate balance of molecule-surface and water-surface interactions. These results should help understand the mechanisms of NCP-mediated HAP crystal growth in bone biomineralization and have broad implications for the design of new peptides and small molecules for biomimetic crystal synthesis.

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Introduction Non-stoichiometric hydroxyapatite (HAP), idealized as Ca10PO4(OH)21, is the main mineral component of bone and dentin. The crystals are in specific structural registry with the main protein component, an insoluble collagen matrix, with which non-collagenous proteins (NCPs) and small molecules are also associated. It is believed that the collagen and NCPs can regulate the nucleation and growth of the HAP phase, from stabilizing multinuclear calcium phosphate clusters in solution to the formation of amorphous nuclei and their transition to crystalline HAP .2-6 The crystallization of HAP could potentially be controlled in a bioinspired manner by a peptide/protein bearing a high binding affinity to HAP similar to the NCPs.7 Furthermore, peptide-HAP interaction can be used for functionalization of the surface of CaP ceramics, which have been broadly applied as dental and orthopedic implants due to their notable biocompatibility with the target tissue.8 Surface functionalization could enhance HAPextracellular matrix recognition to promote the regeneration of damaged tissue.9-10 Synthetic peptides mimicking sequences of natural proteins in the extracellular matrix (ECM) of teeth and bone, for example, fibronectin, osteopontin (OPN) and bone sialoprotein (BSP), provide viable candidates for functionalizing motifs.11-12 Therefore, a comprehensive understanding of the binding mechanism to HAP at the inorganic-organic interface is essential not only to identify the factors controlling the crystal morphology via biomineralization pathway, but also to the design HAP-recognizing peptides. Many of the relevant NCPs are enriched in charged acidic amino acids, such as glutamic acid, aspartic acid and phosphorylated serine12, which are important for surface binding as shown in many studies by means of solid-state nuclear magnetic resonance, quartz crystal microbalance and atomic force microcopy.13-15 Intriguingly, some genetically-engineered peptides (12- to 204


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mers), which contain few acidic residues and bear low negative charge density also exhibit high HAP-binding affinities compared to natural sequences.7, 12, 16 However, these techniques cannot simultaneously provide data for the peptide binding affinity with the associated molecular level structures.17 A range of molecular simulation studies focusing on the peptide structure and binding affinity have been reported previously on many types of surfaces18-24, but simulation of peptide/protein adsorption on surface are severely limited in accessible time scales due to the high conformational entropy in an explicit water solution25-27. In particular, the quantitative evaluation of free energy change upon adsorption presents more difficulties in sufficiently sampling the conformation space of protein/peptide, especially in the vicinity of surfaces28, where the conformation of the adsorbate is severely constrained. A combination of parallel tempering with advanced sampling techniques, such as metadynamics, umbrella sampling and Bennett acceptance ratio, has recently developed to increase the overall sampling efficiency17, 26, 29

. These techniques do, however, require huge computational cost. Simulation of the adsorption of small molecules, such as amino acids and side chain

analogues, provides an alternative way for studying the structure-activity relationship of protein/peptide at the molecular interface by successfully overcoming the sampling problem closely related to the system size.24, 30-33 For example, the adsorption free energies of the side chain groups on rutile titanium surface (100) and (110) faces has been successfully determined in two separate simulation studies30, 33, which shows that the affinities for the surface depended strongly on the details of surface structure. These studies clearly demonstrate that knowledge on the binding propensities of individual sidechain analogues and the related underlying atomiclevel interactions will enable one to modulate the surface binding behaviors of particular residues,

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which are embedded in a peptide. This is particularly true for unstructured proteins, such as the majority of NCPs, which lack secondary structure. Compared to rutile titania, HAP exhibits distinct chemistry and surface structure, featuring ionic groups, Ca2+, PO43- and OH-. The presence of these highly charged ions result in stronger interactions with the interfacial water layers compared to TiO2, with which the peptide/protein must compete to gain access to the inorganic surface. Therefore, it is reasonable to expect a different interaction mechanism underlying the trends of sequence, conformation and binding energy of peptides on HAP, which, in principal, require the identification and characterization of binding affinities of side chains of individual amino acids. We have previously examined adsorption energetics and conformations of whole amino acid molecules34 and small 12-mer peptides17 on HAP surfaces. For amino acid molecules, both side chain and backbone were involved in binding to the surface and only the side chain was involved in the case of peptides. To distinguish between contributions from the side chain and from the terminal amine or carboxy groups, we report in the present study on the interactions and binding energies of side chain analogues (SCAs) where the terminal amine and carboxyl group are absent, and compare the results to those obtained for the complete amino acid molecule. To further distinguish the separate contributions from the building blocks of amino acids, the adsorption free energy of the zwitterionic backbone (BB) is calculated for comparison. For the protein or polypeptide, generally the way to evaluate the role of backbone in the surface adsorption is to model polyglycine. Therefore, in line with polyglycine for the protein/peptide, the backbone model used here for a single amino acid is a glycine molecule. The amino acid residues we investigate here are acidic (glutamate, aspartate), polar, neutral (serine) and phosphoserine at various protonation levels on two HAP surfaces, (100) and (001). To the best of our knowledge, 6


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no systematic potential of mean force (PMF) simulation study has been reported for side chain groups adsorption on HAP surfaces. The results presented here may serve to predict adsorption affinity of peptides on HAP surfaces for controlling crystal growth and potentially improving the design and applications of medical implants.

Simulation Methodology Structure Preparation for Surface Initial Structure and Small Molecule Models Here, we have focused on two of the most significant surfaces of hydroxyapatite, namely the (001) and (100) surfaces, which act as the binding sites for small molecules and proteins/peptides.34 There are two possible terminations for (001) surface and more termination choices for (100) surface.35 In our previous study36, we have carefully investigated the stability of the surfaces with different terminations by calculating the solid-vacuum interface energy per unit surface area (surface energy), and the solid-water interface energy per unit surface area (hydrated surface energy) based on the full surface structure sufficiently relaxed via MD simulation (15 ns). Particularly, two terminations of pure Ca2+ and the combination of Ca2+ and PO43-, are selected for investigations, considering the possible high binding capacity of charged motifs in organic molecules to surface calcium ions. These surfaces are denoted as (hkl)_Ca or (hkl)_Ca-PO4. As reported in other computational studies35, the surface energies in vacuum are often very close. Our results demonstrated that the surface energies for the HAP terminated with Ca2+ are lower than that for the mixed terminations in both (100) and (001) systems and (001)_Ca surface appears to be the most stable surface with the lowest surface energies. The obtained hydrated surface energy was also quite close37-38, except for the (001)_Ca surface, in which a slightly negative interfacial energy of about -58.9 mJ.m-2 was obtained. We observed 7



that the highest coordination number of Ow in water molecules to surface Ca2+ in the (001)_Ca surface leads to the strong electrostatic interactions for the negative energy, while the highest positive interfacial energy (1046.2 mJ.m-2) and the lowest coordination number of Ow to surface Ca2+ were obtained for (001)_Ca-PO4.36 Also the positive hydrated energy for the (100)_Ca surface (324.9 mJ.m-2) was smaller than that for the (100)_Ca-PO4 surfaces (391.2 mJ.m-2). These results may indicate a strong interaction between the (001)_Ca surface and water layers with the similar trend found in other MD simulations and energy minimization calculations.35, 37 With the above observations in mind, two stoichiometric HAP surface structures with terminations of Ca2+ ions, (100)_Ca and (001)_Ca surfaces, were employed for the calculations of adsorption free energy of the side chains. The cleaved crystal slab along the required crystallographic direction is reconstructed to obtain a dipole-moment free surface, by removing half of the atoms from the upper face of the slab to the lower face.34, 39 Specifically, for the HAP (001)_Ca surface, alternate rows of Ca (I) ions along the a axis are removed to the bottom of the slab. The (001)_Ca surface constructed in this way has been widely used in the simulation of the organic molecule-water-HAP systems.35, 40-42 In a similar way, alternate rows of Ca (II) ions along the c axis are removed to the bottom of the slab to generate the HAP (100) surface model.40 An HAP slab with dimensions of 37.7 Å × 37.7 Å × 27.5 Å is generated for the (001) surface and 37.7 Å × 34.4 Å ×32.3 Å for the (100) model surface. Except for the defects induced by removing the ions, we have not considered the surface defects, such as steps, terraces, kinks and so forth, due to the absence of experimental information about the density and distribution of these defects. The selected amino acid for investigation include glutamate, aspartate, phosphoserine and serine. In our previous study we only focused on two types of amino acids interaction with the 8


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HAP surface, namely, phosphoserine (Ser-OPO3) and glutamate (Glu), which are deprotonated with overall charges of -2 and -1, respectively, at physiological pH. Here we extend our interest to two other amino acids (Aspartate, Asp and Serine, Ser), and also consider a protonated state of phosphoserine (Ser-OPO3H). The preparation of these sidechain models are implemented by replacing the α–carbon, amine and carboxy groups by a hydrogen atom as shown in Figure 1a. Molecular Mechanics Force Field The CHARMM22 force field43 and TIP3P model44 are applied to treat the small molecules and explicit water molecules, respectively. These force fields have been widely and successfully used in biological systems in the condensed phase. Force fields for sidechain analogs are generated from CHARMM22 by performing necessary modifications to the side chain β-carbon parameters in order to use the protein force filed parameters for SCA as shown in Figure 1b. The Lennard-Jones (LJ) parameters for modified aliphatic carbon connecting to the backbone (either CH2 or CH3, depending on the amino acid) was taken from the standard CHARMM force field. The partial charge of β-carbon was determined by reducing the original partial charge of this atom by the amount of charge one hydrogen atom takes (in Figure 1b). The bonded parameters (bonds, angles, and torsions), which are already defined in the force filed, are directly used for the resulting connectivity to the added hydrogen atom. This treatment to obtain the force field has been successfully used for calculating the solvation free energy of side chain groups in water.45 The original HAP force field developed by Hauptmann et al.46 is described as a summation of Coulombic and Born-Mayer-Huggins (BMH) potentials for electrostatic and van der Waals based on the rigid model. We have previously successfully converted the original BMH potential function to CHARMM22 function of LJ potential and optimized the LJ 9



parameters in term of the BMH curves.34 A set of rigorous benchmark calculations are performed to systematically examine the performance of the optimized LJ parameters.34 These optimized parameters were then applied to treat the interactions between small organic molecules and HAP crystal faces by using Lorentz-Berthelot mixing rule. In our previous research, we have highlighted the importance of careful benchmarking of classical force fields when employed for interfacial reaction processes, such as biomineralization, involving organic-water-mineral interactions. The calculated interfacial properties were carefully benchmarked to experimental data and to results from density functional theory calculations with explicit consideration of water molecules around the adsorbate plus the Poisson-Boltzmann continuum model for longrange solvation effects. Details about parameter optimization and benchmarking for HAP model surfaces is provided in our previous work.34 Previous DFT calculations found that for the stoichiometric (100) surface, water dissociates at very low coverage47. The classic force field employed here certainly cannot capture the chemical dissociation that may happen on the HAP (100) surface. However, the stoichiometric HAP (100) surface employed in MD simulations with classic force field still provides a suitable surface model to investigate binding affinities of amino acids, peptides and proteins with HAP covered by water layers extending from the bulk region to the interface, as reported in many computational studies.37, 40, 48-49 MD Simulation Details The simulation systems consisting of the HAP (100) or (001) surface and explicit water plus side chain analogs are prepared by introducing the pre-equilibrated bulk water of thickness more than 50 Å on top of the crystal faces. Na+ counter-ions are added to keep the system charge-neutral in the presence of the negatively charged side chain analogs. Isothermal-isobaric ensemble (NPT) simulations (300 K and atmospheric pressure of 1 bar) with periodic boundary 10


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condition in all directions are performed for 20 ns and then NVT ensemble simulations (at 300 K but with constant box volume) for the free energy calculations. In NPT simulations, ParrinelloRahman50 and Nosé-Hoover51-52 are used for controlling the system pressure and temperature, respectively. A time step of 1 fs is used. The particle mesh Ewald method53 is applied to treat electrostatic interaction with a cutoff of 14 Å for the separation of direct and reciprocal space. For van der Waals interactions, a switching scheme is used for interatomic distances between 12.0 and 13.0 Å. Potential of Mean Force Calculation Method The PMF is defined as the free energy along the separation distance between the molecule and each HAP crystal face, and thus has the ability of providing the energetically most favorable position and conformation of the adsorbed molecule at the surface. The umbrella sampling approach54 combined with weighted histogram analysis method55 are applied for the calculation of PMF. Here we provide only a brief description of the calculation method. In the umbrella sampling method, a series of biased simulations are performed at various small molecule-HAP separation values spanning the entire range of interest to construct the PMF. The separation distance along the z-direction (normal to the surface) between the center of mass of the small molecule and the outermost Ca2+ ion layer at each HAP face is defined as the reaction coordinate, which extends from ~2.0-3.0 Å (the adsorbed state) to ~12.0 Å (the desorbed state in the bulk water). Therefore, the constraining potential is only applied to control the translation of the adsorbate in the z-direction and the molecules can move freely in the other two directions (xy-plane) during the umbrella sampling simulations. Through a 20-ns NPT simulation, the initial binding state of the small organic molecule at the surface is obtained. Next, the PULL module in Gromacs 4.0 package is applied to generate a series of structures from the 11



bound state for preparing the initial structure used in window simulations of umbrella sampling. We have also designed a second scheme to construct the initial structures for each window, wherein the free energy calculation started from the desorbed state of the organic molecule in the bulk solution. The umbrella sampling simulations were performed in a successive way with the initial structure for the next window obtained from the preceding sampling simulation. Each simulation window simulation with a gap distance of 0.5 Å between the adjacent windows is performed for 6 ns and the last 4 ns is used for the data analysis. In the vicinity of the surface, the gap distance is set to be 0.1 Å for ensuring the accuracy of the calculated results. Correspondingly, the umbrella harmonic potential constant of 10,000 kJ·mol-1·nm-2 and 80,000 kJ·mol-1·nm-2 are employed. According to our calculations, these two schemes generated highly consistent results, indicating the initial structure has little tiny effect on the results if a careful setup of umbrella sampling is prepared. Results and Discussion Charged SCA adsorption on HAP (100) surface Figure 2 shows the calculated adsorption free energy profiles for all four side chains at the HAP (100) surface. The adsorption free energy profiles of three SCAs (exception is Glu) feature two significant free energy wells separated by a saddle point at ~ 3-4 Å from the surface. The free energy approaches zero beyond 10 Å from the surface, where the SCA is effectively immersed in bulk water. The Ser-OPO3 with two negative charges shows the strongest binding affinity to the surface with a global free energy minimum of about -17.5 kcal·mol-1. The protonated phosphoserine SCA, Ser-OPO2OH, and the Asp SCA with one negative charge each have lower affinities to the surface with minima of -8.9 and -10.8 kcal·mol-1, respectively (Table 1), which is nearly half to what is observed for Ser-OPO3. These results indicate that electrostatic 12


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interaction via the formation of salt bridges between the -COO- group and Ca2+ ions on the surface is the dominating force for adsorption in the present system. The global free energy minima for all three SCAs occur at a distance of ~ 2 Å from the surface, which is within the first hydration layer at the surface consistent with our previous work.17, 34 The conformations shown in Figure 3, corresponding to the global free energy minima, reveal that the preferred binding orientation of all three CSAs is the “standing up” configuration (Figure 3a-c), which favors the formation of salt bridges between –OPO32-, –OPO3OH- or –COOgroup (in Asp) and the outermost Ca2+ ions on HAP surface. It is observed that six salt bridges are formed between Ser-OPO3 and Ca2+ ions, while three and two salt bridges for Ser-OPO2OH and Asp, respectively (Table 2). This observation is consistent with the trend of adsorption free energies obtained in the PMF profiles (Figure 2), thus further confirming that electrostatic interactions dominate the SCA-surface interactions. The favorable conformation of SCAs on the surface is largely influenced by the strong affinity with the substrates. In contrast to the HAP surface, SCAs are oriented parallel to TiO2 surfaces and the binding energies are -0.2 ~ -0.6 kcal.mol-1, due to weak dipole-charge interactions originating in the Ti and O surface layers.33 We pay special attention to the adsorption free energy of Glu, which has one negative charge, but did not exhibit a strong binding affinity to the HAP (100) surface. The free energy profile features a minimum of only -3.3 kcal·mol-1 obtained at a separation of ~ 4.0 Å from the surface (Figure 2d) and a weaker well of about -1.2 kcal·mol-1 at ~ 5Å. For the free energy Glu adsorption, we performed additional three sets of window simulations from 2.0 to 6.0 Å (each set includes 18 windows) with different initial configurations to ensure that the conformational space is accurately sampled in the vicinity of solid surface and highly consistent results are obtained. We noticed that the “standing up” conformation on the (100) surface also resulted in 13



two salt bridges (Figure 3d), demonstrating that the -COO- group of Glu side chain has penetrated the water layer. Compared to the other SCAs, however, the location of the global free energy minimum is farther away from the surface (at ~ 4.0 Å) and no favorable binding site in the region closer to the surface is observed (Figure 2). This is reasonable considering the larger size of Glu’s non-polar hydrocarbon chain compared to Asp or Ser. Approaching the surface any closer requires the breaking of the already-formed salt bridge to allow the adjustment of the molecule’s orientation with respect to the surface. This is a thermodynamically unfavorable process with a free energy barrier of about 6 kcal/mol due to the strong electrostatic interactions from salt bridges. This unfavorable process actually reflects the loss in entropy as a result of the conformational restraining imposed by the surface on the SCA. This effect becomes relatively more pronounced as the molecule becomes larger. Additionally, an SCA of larger size needs to displace more waters for fitting into the vacated surface site. While this may increase the entropy of the released water molecules, the enthalpic benefit from bringing the charged SCA closer to the charged surface is apparently not sufficient to compensate.33 Since the HAP surfaces used here are terminated with the Ca2+ ions, which are the potential binding sites for charged molecules, the specific distributions of outermost Ca2+ would influence the binding of small molecules with the surface. As discussed above, multiple salt bridges between –OPO32-, –OPO3OH- or –COO- group and the outermost Ca2+ ions on HAP (100) surface are formed. Therefore, the defects on HAP (100) surface created while generating the dipole moment-free surface would influence the exact binding sites of small molecules,35 which relies on the specific distributions of the surface Ca2+.

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Charged SCA Adsorption on HAP (001) Surface The HAP nanocrystals in bone and dentine have a large (100) face and a small (001) face indicating that NCP-mediated HAP crystal growth is fastest in the direction perpendicular to the (001) face.56 This may ascribed to weak interactions between the adsorbing molecules and the HAP (001) face.34 The free energy landscapes of SCAs on HAP (100) and (001) face are shown in Figure 4. Compared to the (100) face, the adsorption PMF profiles on (001) generally display less complexity with only one free energy minimum. As SCAs approach the surface, the adsorption free energies monotonously decrease before attaining the global minimum of ~2 kcal·mol-1 for the monovalent SCAs and ~-5.5 kcal·mol-1 for Ser-OPO3, at a distance of ~3.8-4.0 Å. This is beyond the first water layer on the surface, and hence no free energy barrier exists during the binding process (Figure 4). The free energy minima observed on the (001) surface are much smaller than that on the (100) surface due to shielding effect of the bound waters on the electrostatic interactions, leading to weaker interactions. In our previous study,36 we observed the stronger interaction of water with (001) than (100) arising from the higher coordination number of water molecules to Ca2+ on the (001) surface. Again, the Ser–OPO3 SCA possessing two negative charges exhibits the strongest binding affinity with the surface. The three monovalent SCAs (Ser–OPO3OH, ASP and Glu) have quantitatively identical interaction strength in spite of the difference in their molecular size. This indicates that the molecular size has negligible effect on interactions between SCAs and surface on the (001) surface. In summary, the (001) face behaves differently from the (100) surface because of weaker interactions between water and the (100) surface compared to the (001) surface.36 The corresponding conformations at the free energy minima further confirm, that the SCAs do not significantly penetrate into the structured water layers at the (001) surface (Figure 15



5). This leads to failure of salt bridge formation between any of the SCAs examined and the (001) surface (Table 2), although SCAs still prefer the standing up conformation on the surface, similar to results for (100). Therefore, the binding of adsorbates on the surface shows weak dependence on the distributions of the outermost Ca2+. The comparison of Glu side chain adsorption on HAP (100) and (001) surfaces clearly demonstrates that the impact of side chain size only occurs in cases where the molecule is capable of penetrating into the water layers. Our results indicate that the binding affinity of SCAs is strongly depends on the structural characteristics of the crystal surface, which dominates the ability of adsorbents penetrating into the water layer.36 Furthermore, the surface-bound water plays a crucial role in mediating the adsorbents and surface and thus influences the adsorption behavior.19, 36 These results highlight the signal importance of proper benchmarking of interfacial terms in MD FFs.25, 34, 36 Charge-neutral SCA and BB Adsorption on HAP (100) and (001) Surfaces Since amino acids are composed of side chains and backbone, it is of great interest to investigate the adsorption free energy of the backbone, as a charge-neutral though zwitterionic group, which may provide an alternative way to reveal the role of charged side chains in controlling amino acid and peptide adsorption. The uncharged serine sidechain is selected for comparison with the zwitterionic BB (Figure 1). The free energy profile of the Ser SCA shows unfavorable adsorption energies at both (100) and (001) surfaces (Figure 6 a, b). Both surfaces generally show sustained resistance against Ser SCA adsorption, leading to a large free energy barrier, which arises from the interfacial water layer. Thus the Ser SCA is actually driven away from the interfacial region by both surface models studied here. Similar adsorption behavior of Ser SCA is also reported on negatively charged titania (110) surface constructed by attaching 18 hydroxyl groups, which displays repulsive or 16


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comparatively weak interaction with the adsorbent.31 However, Brandt et al.33 found that serine side chain is the strongest binder with TiO2 (100) surface among the SCASs of 20 naturally occurring amino acids and the negatively charged SCAs (Asp and Glu) show relatively weaker interaction with the TiO2 (100) surface. The difference in SCA adsorption behavior at HAP and TiO2 (110) surfaces indicates that the specific surface structure and the surface-bound water layers play a critical role in controlling the affinities of molecules at the surface. Compared to TiO2, the HAP crystal possesses higher surface charge density because of ionic groups and hence interacts with water more strongly, resulting in the formation of more stable water layers. Therefore, the uncharged Ser SCA showed significantly repulsive interaction with both of the HAP faces studied here. These studies successfully identified the importance of electrostatic interaction (formations of salt bridges between the -COO- group and outermost Ca2+ ions, and hydrogen bonds between the -NH3+ and PO43- surface ions) and surface-bound water layer, in peptides binding with inorganic surfaces.36, 57 In contrast, though the BB is also electrically neutral, the free energy landscape exhibits similar features to those for the charged SCAs because the BB is zwitterionic. Moreover, beyond our expectation the BB shows even stronger interaction with both surfaces than the monovalent SCAs (Figure 6). The binding free energy of the BB is ~-12 kcal.mol-1 and ~ -4 kcal.mol-1 on the (100) and (001) surface, respectively, and approach within ~2-3 Å of the surface. This contrast between zwitterionic BB and monovalent charged SCAs is more pronounced on the (001) face than on (100) (Table 1). The analysis of binding conformations clearly displays, that the BB adopts parallel orientation with respect to the surface, leading to formation of four salt bridges between the COO- group and Ca2+ ions on the surface, and two hydrogen bonds between the -NH3+ and PO4317



surface ions. (Figure 7, Table 2). The significant difference in the free energy profiles for zwitterioninc BB and uncharged serine SCA, both of which are net charge-neutral, demonstrates that the number of charged motifs within the structure actually plays a more critical role in determining their binding affinity with the surface than the net charge. A similar result was obtained in our previous work on adsorption of 12-mer peptides binding on HAP surfaces.36 It is of great interest to investigate whether the free energy sum of individual BB and SCAs is similar to that of the whole amino acid. Adding the free energy values at the position of global minima in PMF profiles of the BB with either Ser-OPO3 or Glu side chains results in -8.5 and 5.3 kcal/mol, respectively, at the (001) surface. Both these values are around two times greater in magnitude than that obtained for the adsorption of the whole amino acid molecule.34 A similar outcome is obtained for the HAP (100) surface. This is puzzling at first because of the greater number of charged motifs existing in the whole amino acid molecule. However, the increase in the size of the molecule results in larger entropic loss due to conformational constraining near the surface,

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which is thermodynamically less favorable for adsorption than

the individual backbone and SCA fragments. Thus, the effect of size on balancing entropy and enthalpy holds whether with respect to BB or the whole molecule. Implications for Peptide/protein Binding in Biomineralization Considering the fact that the HAP binding domains of acidic NCPs are normally negatively charged at physiological pH, the number of charged residues is observed to direct the binding of NCPs involved in bone biomineralization59, which is well explained via the interaction mechanism of SCAs revealed here. One important result from the present work is that the charged functional groups, commonly occurring in proteins that bind to HAP surfaces have favorable interaction with the HAP surface. Independent of their identities, the binding affinity 18


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of SCAs to the HAP (100) surface is larger than that of (001) surface, which is consistent with the behavior of whole amino acid molecule adsorption34. On HAP (100), the binding affinities of the small molecules studied here are predicted to be “Ser–OPO32- > zwitterionic backbone0 > Asp- > Ser–OPO2OH- >Glu- >>> Ser0. Thus binding is mainly dominated by the total number of charges present on the small molecule as well as the molecule size. For charged SCAs, the binding energy also depends on the net charge. Thus, SCAs bind to HAP surface through electrostatic forces, which is in agreement with recent MD simulations of adsorption NCPs at HAP and calcium oxalate surfaces.17, 36, 59 The stronger affinities of Asp and Ser-OPO2OH versus Glu on the (100) face, all of which are monovalent, suggests that a small-sized side chain involved in a protein or peptide may, indeed, facilitate its binding with HAP. One can speculate that proteins with high fractions of shorter charged side chains would have higher binding affinity to HAP surfaces. Our simulations elucidated the important contribution of interfacial water layers in controlling the thermodynamics of the adsorption, as indicated by the repulsive force against the serine approaching both HAP surfaces as well as the weak binding of the monovalent SCAs with the (001) surface. A delicate balance between the adsorbent-surface and adsorbent-water interactions ultimately determines the relative adsorption free energies, which underlies adsorption behavior in the solid-solution interface. Conclusions We performed molecular dynamics simulations to obtain adsorption free energies of SCAs of acidic and uncharged polar amino acids as well as the corresponding zwitterionic backbone fragment, and compared the results to those for the entire amino acid molecule. Binding free energies depend on the number of charged groups rather than simply on net charge. 19



Secondly, it is more difficult for larger molecules to adsorb because of less favorable enthalpic and entropic contributions required to penetrate the tightly bound surface water layer. Our study provides an atomic-level understanding on the peptide/protein-HAP interactions, which may guide the rational design of bioinspired materials in orthopedic and dental tissue engineering.

Acknowledgments This work is financially supported by National Natural Science Foundation of China (No.21606122) and Natural Science Foundation of Jiangsu Province (No. BK20160995) to Z. X. Finical support was also provided by start-up funds from University of Akron and NSF DMR Biomat Grant 0906817 to N.S. Computation resources are provided by the Ohio Supercomputer Center under grant PBS0286 to N. S and Z. X.

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30. Sultan, A. M.; Hughes, Z. E.; Walsh, T. R. Binding Affinities of Amino Acid Analogues at the Charged Aqueous Titania Interface: Implications for Titania-Binding Peptides. Langmuir 2014, 30, 13321-13329. 31. Monti, S.; Walsh, T. R. Free Energy Calculations of the Adsorption of Amino Acid Analogues at the Aqueous Titania Interface. J. Phys. Chem. C 2010, 114, 22197-22206. 32. Pan, H.; Tao, J.; Xu, X.; Tang, R. Adsorption Processes of Gly and Glu Amino Acids on Hydroxyapatite Surfaces at the Atomic Level. Langmuir 2007, 23, 8972-8981. 33. Brandt, E. G.; Lyubartsev, A. P. Molecular Dynamics Simulations of Adsorption of Amino Acid Side Chain Analogues and a Titanium Binding Peptide on the Tio2 (100) Surface. Langmuir 2015, 119, 18126-18139. 34. Xu, Z.; Yang, Y.; Wang, Z.; Mkhonto, D.; Shang, C.; Liu, Z.-P.; Cui, Q.; Sahai, N. Small Molecule-Mediated Control of Hydroxyapatite Growth: Free Energy Calculations Benchmarked to Density Functional Theory. J. Comput. Chem. 2014, 35, 70-81. 35. Filgueiras, M. R. T.; Mkhonto, D.; Leeuw, N. H. d. Computer Simulations of the Adsorption of Citric Acid at Hydroxyapatite Surfaces J. Cryst. Growth 2006, 294, 60-68. 36. Zhao, W.; Xu, Z.; Yang, Y.; Sahai, N. Surface Energetics of the Hydroxyapatite Nanocrystal–Water Interface: A Molecular Dynamics Study. Langmuir 2014, 30, 1328313292. 37. Leeuw, N. H. d. Computer Simulations of Structures and Properties of the Biomaterial Hydroxyapatite. J. Mater. Chem. 2010, 20, 5376-5389. 38. Fabio Chiatti; Massimo Delle Piane; Piero Ugliengo; Corno, M. Water at Hydroxyapatite Surfaces: The Effect of Coverage and Surface Termination as Investigated by All-Electron B3lyp-D* Simulations. Theor. Chem. Acc. 2016, 135, 54. 24


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FIGURE CAPTIONS Figure 1. (a) Atomic structures of the selected sidechain analogs and residue backbone used in adsorption free energy calculations at the water-HAP interface. (b) Comparison of partial charges in CHARMM22 force field for serine side chain shown as an example and the CHARMM22-derived parameters for the SCA used in this study. Only the β-carbon atom changes in charge. Figure 2. Free energy profiles for SCAs on HAP (100) surface. (a) Ser-OPO3, (b) Ser-OPO2OH, (c) Asp and (d) Glu. Figure 3. Binding configurations of SCAs on HAP (100) face at the global free energy minima shown in Figure 2. The side chains form direct binding interactions with the surface by penetrating into the interfacial water layers (a, b). Legend: H = white; C = grey; N = dark blue; O = red; P = tan; Ca = cyan spheres. Water molecules and Na+ ions are omitted for clarity. Figure 4. Free energy profiles for the sidechains of Ser-OPO3, Ser-OPO2OH, Asp and Glu on HAP (001) surfaces. Figure 5. Binding configurations of SCAs on HAP (001) face at the global free energy minima as shown in Figure 4. The side chains indirectly interact with (001) surface through stable interfacial water layers, which sandwiched between sidechains and (001) surface, as shown in (a) and (b). See Figure 3 for color legend. Bulk water molecules and Na+ ions are omitted for clarity. Figure 6. Adsorption free energy profiles for serine SCA (top panel) and amino acid BB (bottom panel) on HAP (100) (a, c) and (001) surfaces (b, d). Figure 7. Binding configurations of serine side chains and backbone on HAP (100) and (001) faces. Water molecules and Na+ ions are omitted for clarity. See Figure 3 for color legend.

28


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Table 1: The adsorption free energy (kcal.mol-1) for residue backbone and side chains of SerOPO3, Ser-OPO2OH, Glu, Asp and Ser.

Side chain (charge) Ser-OPO3 (-2) Ser-OPO2OH (-1) Glu (-1) Asp (-1) Ser (0) Backbone (0)

HAP(001) -5.00 (-4.5)* -1.82 -1.80 (-2.2)* -1.78 +2.00 -3.50

*

HAP(100) -18.20 (-9.80)* -8.89 -3.29 (-8.2)* -10.77 +1.79 -11.93

The values shown in the bracket are the adsorption free energies of the corresponding amino

acids obtained from ref. 34.

29



Page 30 of 38

Table 2: Number of the ion bridge formed between the O atom in the sidechain and the Ca2+ ions on the surface. Side chain

HAP(001)

HAP(100)

Ser-OPO3 Ser-OPO2OH Ser Glu

0 0 0 0

6 3 2 2

Asp Backbone

0 0

4 4

30


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TOC Graphic

31



Figure 1. (a) Atomic structures of the selected sidechain analogs and residue backbone used in adsorption free energy calculations at the water-HAP interface. (b) Comparison of partial charges in CHARMM22 force field for serine side chain shown as an example and the CHARMM22-derived parameters for the SCA used in this study. Only the β-carbon atom changes in charge. 165x75mm (300 x 300 DPI)


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Figure 2. Free energy profiles for SCAs on HAP (100) surface. (a) Ser-OPO3, (b) Ser-OPO2OH, (c) Asp and (d) Glu. 239x175mm (300 x 300 DPI)



Figure 3. Binding configurations of SCAs on HAP (100) face at the global free energy minima shown in Figure 2. The side chains form direct binding interactions with the surface by penetrating into the interfacial water layers (a, b). Legend: H = white; C = grey; N = dark blue; O = red; P = tan; Ca = cyan spheres. Water molecules and Na+ ions are omitted for clarity. 160x127mm (300 x 300 DPI)


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Figure 4. Free energy profiles for the sidechains of Ser-OPO3, Ser-OPO2OH, Asp and Glu on HAP (001) surfaces. 239x175mm (300 x 300 DPI)



Figure 5. Binding configurations of SCAs on HAP (001) face at the global free energy minima as shown in Figure 4. The side chains indirectly interact with (001) surface through stable interfacial water layers, which sandwiched between sidechains and (001) surface, as shown in (a) and (b). See Figure 3 for color legend. Bulk water molecules and Na+ ions are omitted for clarity. 160x143mm (300 x 300 DPI)


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Figure 6. Adsorption free energy profiles for serine SCA (top panel) and amino acid BB (bottom panel) on HAP (100) (a, c) and (001) surfaces (b, d). 239x175mm (300 x 300 DPI)



Figure 7. Binding configurations of serine side chains and backbone on HAP (100) and (001) faces. Water molecules and Na+ ions are omitted for clarity. See Figure 3 for color legend. 159x127mm (300 x 300 DPI)


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Free Energy Calculations of Amino Acid Side Chain Analogues

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