On the Function of Saccharides during the Nucleation of Calcium

Article pubs.acs.org/crystal

On the Function of Saccharides during the Nucleation of Calcium Carbonate−Protein Biocomposites Patrick Duchstein,† Rüdiger Kniep,‡ and Dirk Zahn*,† †

Lehrstuhl für Theoretische Chemie/Computer-Chemie-Centrum Friedrich-Alexander-Universität Erlangen-Nürnberg, Nägelsbach Str. 25, D-91052 Erlangen, Germany ‡ Max-Planck Institut für Chemische Physik fester Stoffe, Nöthnitzer Str. 40, D-01187 Dresden, Germany ABSTRACT: The molecular mechanism of calcium carbonate nucleation in the presence of various types of collageneous proteins is unravelled from computer simulation of ion-by-ion association steps. Single calcium ions are incorporated in the triplehelix by formation of salt bridges to carbonyl and hydroxyl groups of collagen, while single carbonate ions tend to bind laterally to the biomolecule. However, upon multiple ion association, the self-organization of the forming aggregate strongly depends on the triple-helical collagenous strand. In absence of glycosylated lysine residues, we observed that carbonate ions bind to calcium ions that are already incorporated into the triple helix and eventually cause the unfolding of the protein. On the other hand, otolin1, a specific, collagen-like protein found in biogenic calcite-based composites such as otoconia, comprises a particularly high degree of glycosylated amino acids which avoid such “destructive” calcium−carbonate contacts by providing alternative association sites more lateral to the backbone. This leads to the formation of a saccharide−calcium carbonate agglomerate that does not compromise the protein’s triple helix and constitutes the organic−inorganic interface of the nucleating biocomposite.

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hydroxylated, and of those, 97% become glycosylated.8 While structurally very similar, otolin-1 contains an even larger content of lysine residues and may thus be expected to exhibit a particularly high degree of glycosylation. Otolin-1 is a collagenlike protein specific to calcite-based biominerals such as otoconia (a biomineral being part of the acceleration sensors in the inner ear). While there is a large body of experimental evidence related to the importance of saccharides for inducing ion aggregation, we lack mechanistic knowledge of how this process and its interplay with the nucleation and growth of a hierarchical composite actually works. To look into the molecular scale mechanisms, computer simulations have proven a powerful tool of investigation. In what follows, we build on simulation models and algorithms developed earlier and provide a direct comparison of ion association to a small series of collagen models. Such molecular models cannot account for the full complexity of the organic matrix, yet they allow a focus on specific aspects of the biomolecules and are, thus, particularly suited to elaborate detailed mechanistic insights.

INTRODUCTION Bone, teeth, and otoconia (functional biominerals of the human body) are composite materials, comprising an inorganic phase which closely interacts with organic tissue, in many cases collagen.1−5 These fiber proteins are constituted by a triplehelical backbone which, as a simple approximant, can be mimicked by a (Hyp-Pro-Gly)n polypeptide. However, biogenic collagen obtained from biominerals is known to comprise of more complex (X-Y-Gly) sequences giving rise to an analogous triple helix but offering a variety of side groups along the protein backbone. The most prominent feature of this more lateral part of collagen is given by saccharide groups. From NMR experiments, saccharides have recently been identified as the dominant part of the organic−inorganic interface in bone and teeth.6 While the explicit function of these residues remained unknown so far, it is intuitive to assume that millions of years of evolution did not place the saccharide groups coincidentally. This idea is supported by the identification of different saccharide contents in different types of biominerals. Glycosylation of collagen fibers takes place in the endoplasmatic reticulum, before triple helices are formed. Lysine becomes hydroxylated by lysyl hydroxylase, and then glycosylated by β-(1-0)-galactosyl- and α-(1-2)-glucosyltransferase, resulting in α-D-glucopyranosyl-β-D-galactopyranosylhydroxylysine (GGH).7 The degree of glycosylation of collagen fibers varies with its type and location of synthesis. For example, in collagen type X, 65% of the lysine residues become © XXXX American Chemical Society

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MODELS AND METHODS

To explore the role of saccharide side groups in collagen-based biominerals, we transfer a recently presented molecular simulation Received: July 15, 2013 Revised: September 10, 2013

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dx.doi.org/10.1021/cg401070h | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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Figure 1. (a) Association of a single calcium (yellow, left) and a single carbonate (gray/green, right) ion to nonglycosylated collagen in aqueous solution (15.000 water molecules, not shown). The purple ribbons indicate the backbone of the triple-helical peptide. (b) The association of several calcium (yellow) and carbonate (gray/green) ions to the nonglycosylated collagen leads to distortion and finally to the unfolding of the collagen triple helix. For clarity, solvent molecules (15.000 H2O) are not shown.

Figure 2. Calcium and carbonate ion association to the glycosylated otolin model in aqueous solution (solvent not shown). The inset at the right shows the coordination of a carbonate ion by hydrogen bonding to an amino group (left) and to several saccharide residues (top, bottom, and right). This induces the formation of calcium carbonate clusters laterally bound to otolin without compromising its triple-helical structure (see also Figures 1 and 3).

used to describe the atomic interactions of calcium carbonate,10 water,11 and the biomolecules. 12,13 The molecular dynamics simulations are based on a recently developed atomistic simulation

protocol mimicking ion-by-ion association and self-organization during apatite−collagen nucleation9 to the aggregation of calcium carbonate− collagen composites. In full analogy to ref 9, empirical potentials are B



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Figure 3. Calcium carbonate aggregation promoted by glycosylated otolin. The same configuration is shown at two representations demonstrating the intact triple helix (left) and the intergrowth of ionic clusters and the saccharide residues (right, highlighted in blue). scheme for investigating crystal growth from solution.14 The KawskaZahn approach reflects an iterative procedure for tackling nucleation from very dilute solutions mimicking ion diffusion to the aggregate by a docking procedure and investigating aggregate growth and relaxation from simulated annealing molecular dynamics simulation runs after each ion association step.15 On the basis of three independent growth runs, we investigate the association of up to 200 ions to three different collagen models of different complexity. Similar to previous simplifications of collagen models,9 the triple-helical backbone of human Otolin-1 and collagen-X were mimicked by a subsequence, residues 176−208 of uniprot accession no. A6NHN0 and 389−421 residues of P23206, respectively. The collagenous domain was assumed to be a parallel homo trimer and all lysine residues in position Y of GLY|CYS-X-Y were replaced by GGH.

increasingly dramatic, during the association of further ions (middle) until the helical structure is finally disrupted (right). Arguably, this finding could be related to the strong simplification of our collagen model, relying on a (Gly-ProHyp)n polypeptide approximant, rather than on collagen species, as actually present in the vestibular system.16 We thus repeated our aggregate growth simulations in order to investigate the interplay of otolin-1 with calcium and carbonate ion association. Indeed, for glycosylated otolin-1, the picture is substantially different: Figure 2 shows that the disaccharide chains and the protonated amine groups of lysine offer particularly favorable association sites for carbonate ions. Favorable hydrogen bonding provided lateral to the triple helix acts as a selective shield for CO32− ion association. While single Ca2+ ions may still be incorporated inside the triple helix, no ion pairs, triples, etc. are formed within the “interior” of otolin-1, thus keeping the triple-helical structure intact. The nucleation of Cax(CO3)y2x−2y aggregates instead occurs at the contact regions of disaccharide side chains and the aqueous solution. Upon formation of larger ion agglomerates, this leads to the intergrowth of (largely disordered) calcium carbonate with the glyclosylated side chains of otolin. Figure 3 illustrates this phenomenon for a later stage of aggregate growth (comprising 184 ions), clearly indicating the intact triplehelical structure of the protein. To support this mechanistic concept, we performed further aggregate growth simulations using a nonglycosylated model of the otolin-1 sequence described above. For this “control experiment”, it is indeed observed that calcium carbonate ion clusters form in between the collagen strands and finally disrupt the triple helix in full analogy to the (Gly-Pro-Hyp) n polypeptide approximant, as discussed earlier. Even stronger evidence was collected from comparing two independent simulation runs dedicated to calcium carbonate and hydroxyapatite aggregation to glycosylated collagen-X. The corresponding triple helix exhibits a smaller degree of glycosylation (∼3% of all amino acids) than otolin (∼12 % of all amino acids). Strikingly, glycosylated collagen-X was found to induce calcium phosphate association and the nucleation of apatite motifs in full analogy to the (Gly-Pro-Hyp)n polypeptide approximant, while calcium carbonate aggregation still leads to disintegration

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RESULTS To allow direct comparability to a previous study of apatite− collagen composite nucleation, we initially adopted the simple (Gly-Pro-Hyp)n collagen model from ref 9. Indeed, the association of a single calcium or carbonate ion to this nonglycosylated collagen triple helix was found in full analogy to calcium and phosphate ion association: the Ca2+ ions are incorporated into the helical center, form salt-bridges with carbonyl and hydroxyl groups and thus stiffen the triple-helical structure, as shown in Figure 1a. On the other hand, the larger PO43− and CO32− ions attach laterally to the helix. A striking discrepancy of apatite-(Gly-Pro-Hyp)n and calcium carbonate-(Gly-Pro-Hyp)n composite nucleation is observed upon further ion association. The simulations related to apatite showed that the phosphate ions remain at lateral positions, while the incorporation of [Ca3F] motifs into the collagen backbone keeps the overall structure of the triple helix intact. In contrast to this successful interplay of apatite motifs and biomolecular structure, the association of calcium and carbonate ions leads to increasing unfolding of the triple helix. Calcium ions that are incorporated in the (Gly-Pro-Hyp)n triple helix tend to drag carbonate ions from the otherwise lateral association site. As a consequence, Cax(CO3)y2x−2y aggregates are formed within the collagen backbone, thus pushing the peptide strands apart. Figure 1b illustrates this process as observed for the CaCO3 ion pair (left), and C



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to guarantee the structural integrity of the fibrous collagen macromolecules. In case the triple-helices are not or only insufficiently glycosylated, the scenario of collagen disruption is the preferred reaction mechanism. With this, the first and basic insight is given for an open problem which was provided already in 1996,21 and which is still pending,18 the function of polysaccharides in biomineralisation. More recent investigations in biogenic and biomimetic otoconia (calcite-based biominerals in the inner ear of vertebrates) revealed the presence of parallel arrangements of undistorted (linear) fibrils of the organic component within the composite system. 22,23 The biomimetic otoconia have preferably been grown by double-diffusion in gelatin-gel matrices, which, at first glance, seems to be contradictory to our present calculations, showing the destructive force of calcium carbonate during attachment at/in nonglycosylated collagen. However, commercial gelatin still contains 0.5−1.0 wt. % of covalently bound saccharides,24 which are not detached during gelatin processing from collagen and which help for calcium carbonate nucleation without disruption of the triplehelices structure. Despite the enormous complexity intrinsic to biogenic systems, molecular simulations dedicated to selected aspects of biomineral formation may provide mechanistic insights at a unique level of detail.15 On this basis, at least a qualitative understanding of composite nucleation, the development of hierarchical structures, and the resulting materials properties are within reach.9,25 Here, we show that disaccharide groups attached to collagen represent a crucial modification, or better to say, functionalization of the biomolecule. In absence, or at insufficient different degree of glycosylated lysine residues, collagen was observed to unfold during calcium carbonate based composite formation, while otolin-1 comprises a sufficient amount of ‘shielding’ disaccharide groups to ensure structural integrity of the triple-helix. Moreover, our simulations show that the organic−inorganic interface of the forming composite is constituted by the intergrowth of glycosylated collagen and largely disordered calcium carbonate.

of the protein at late stages of aggregate growth (as compared to nonglycosylated collagen). Accordingly, collagen glycosylation plays a minor role for calcium phosphate-based composite formation but is of such critical relevance to carbonate association that a specialized collagen type, the heavily glycosylated otolin, is needed for structural integer carbonatebased composites. The comparison of the different mechanisms of apatite association to collagen and calcium carbonate association to otolin furthermore hints at a different structural interplay. The hierarchical nature of apatite−collagen composites could be rationalized by apatite motif orientation induced by collagen, namely, by correlation of the crystallographic c axis of apatite and the long axis of the protein fiber9 and by the generation of intrinsic electric fields taking over control of further hierarchical developments.17 In contrast to this, the disaccharide side chains of otolin provide much less structural ordering to the forming calcium carbonate aggregates. The analysis of the distances of

Figure 4. Occurrence profile calculated for Ca···O distances within the (largely disordered) ion clusters associated to otolin (solid curve) (see also Figure 3). The dashed lines indicate the corresponding distances as characteristic for the calcite and aragonite crystal structures. Taking into account Ca2+ contacts to oxygen atoms of CO32− groups, saccharide hydroxy groups, and water molecules, the averaged coordination number of the organic−inorganic interface is 6.97, while the coordination numbers of calcium by oxygen in the crystal structures of calcite and aragonite are 6 and 7, respectively.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

Ca2+−O (from CO32−) contacts and coordination numbers averaged over the full aggregate growth trajectory (Figure 4) reveals only weak ordering. Thus, the very first nucleation step can be described as the formation of areas of largely disordered (“amorphous”) calcium carbonate, a metastable state which is often discussed as a precursor phase for the formation of calcium carbonate-based biominerals.18−20 The actual formation of calcite, which is expected at much later stages of aggregate growth, is not observed on the basis our simulations, which are limited to the infancy of nucleation. However, this does not rule out a putative role of the biomolecule during metastable−stable phase transformations of more mature calcium carbonate states.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge support from the DFG Grant ZA420-7 and the Erlangen cluster of excellence “Engineering of advanced materials”.

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REFERENCES

(1) Biomineralisation: Cell Biology and Mineral Deposition; Simkiss, K., Wilbur, K. M, Eds.; Academic Press: San Diego, 1989. (2) Biomineralisation: Progress in Biology, Molecular Biology and Application; Bäuerlein, E., Ed.; Wiley-VCH: Weinheim, Germany, 2004. (3) Handbook of Biomineralization: Biological Aspects and Structure Formation; Bäuerlein, E., Ed.; Wiley-VCH: Weinheim, Germany, 2007. (4) Addadi, L.; Weiner, S. Angew. Chem. 1992, 104, 159−176; Angew. Chem., Int. Ed. Engl. 1992, 31, 153−169. (5) Biomineralisation and Biological Metal Accumulation; Westbroek, P., de Jong, E. W., Eds.; D. Reidel Publishing Company: Dordrecht, NL, 1983.

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CONCLUSIONS For the nucleation of calcium carbonate-based biominerals, the glycosylation of collagen seems to be an important biological benefit. The simulation results clearly show that saccharide groups attached to collagen triple helices not only strongly interact with calcium and carbonate ions but also are necessary D



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On the Function of Saccharides during the Nucleation of Calcium

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