Osteocalcin facilitates calcium phosphate ion complex growth as revealed by free energy calculation

Abstract

The nanoscopic structural and thermodynamic basis of biomolecule-regulated assembly and crystallization of inorganic solids have a tremendous impact on the rational design of novel functional nanomaterials, but are concealed by many difficulties in molecular-level characterization. Here we demonstrate that the free energy calculation approach, enabled by combining advanced molecular simulation techniques, can unravel the structural and energetic mechanisms of protein-mediated inorganic solid nucleation. It is observed that osteocalcin (OCN), an important non-collagenous protein involved in regulating bone formation, promotes the growth of nanosized calcium phosphate (CaP) ion clusters from a supersaturated solution. Free energy calculation by umbrella sampling indicates that this effect by OCN is prominent at the scale of 1 to 3 nm ion-association complexes (IACs). The binding interactions between gamma-carboxyl glutamate and the C-terminal and, interestingly, the arginine side chains of OCN and IACs stabilize under-coordinated IACs, thus promoting their growth. The promoter effect of OCN on the enlargement and further aggregation of IACs into cluster assemblies of tens of nm are confirmed by conventional molecular dynamics simulation and dynamic light scattering experiments. To the best of our knowledge, this is the first time that the free energy landscape of the early stages of CaP nucleation is shown. The free energy change as a function of IAC size shares the feature of decreasing monotonically as shown previously for the calcium carbonate system. Therefore, the nucleation of both these major biominerals apparently involves an initial phase of liquid-like ionic aggregates. The structural and thermodynamic information regarding OCN-CaP interactions amplifies the current understanding of biomineralization mechanisms at the nanoscale, with general relevance to biomolecule-tuned fabrication of inorganic materials.

The structural role of osteocalcin in bone biomechanics and its alteration in Type-2 Diabetes

“…While OC is a 49-residue protein (see Fig. 1 a), previous molecular dynamics simulations have generally considered amino-acids 13–49 based on the structure (code 1Q8H) available in the Protein Data Bank (PDB) 37 , 38 . This study considers the full OC protein by using the Molefacture plugin of the Visual Molecular Dynamics (VMD) package 39 to build the remaining portion of the protein which exists as a random coil segment 40 .…”

The Thermodynamics of Medial Vascular Calcification

“…Recent analyses of the early stages of CaP precipitation using high-resolution molecular techniques and ab initio molecular dynamics simulations (Lin and Chiu, 2018;Zhao et al, 2018) provide better insights into the molecular processes involved in CaP precipitation (Habraken et al, 2016). During the initial step, calcium and phosphate ions associate to form dinuclear and trinuclear complexes and subsequently polynuclear Posner clusters (Beniash et al, 2000) composed of nine Ca 2+ ions and six phosphate PO 4 3− anions, surrounded by 30 water molecules, as depicted in Figures 2A, 3.…”

“…Figure 4A shows the typical Ca 2+ligand groups in biomolecules. In descending order according to their binding strength, these groups include phosphonate, phosphate > carboxylate > sulfonate = sulfate > alkoxide ≈ water (Zhao et al, 2018). Several kinds of biomolecules may contain large numbers of these groups, as it is the case of phosphate in multiphosphorylated proteins (Figure 4Aa), such as phosvitin (Greengard et al, 1964), involved in physiological calcifications (Sarem et al, 2017).…”

Injectable Enzymatically Hardened Calcium Phosphate Biocement

“…It is known that the IGF1 factor stimulates the type I collagen synthesis, osteocalcin production and alkaline phosphatase activity [64,65]. On other hand, the osteocalcin (OC) has been found to accelerate nucleation by promoting the growth of nanosized calcium phosphate ion clusters and suppressing hydroxyapatite crystal growth [66,67] as well as osteopontin (OP) in mixture with osteocalcin-promoted hydroxyapatite formation [68]. Both proteins are induced in the mineralization stage and are characterized by mature osteoblasts [69].…”