Control of Calcium Oxalate Crystal Growth by Face-Specific Adsorption of an Osteopontin Phosphopeptide

Abstract

Mineral-associated proteins have been proposed to regulate many aspects of biomineralization, including the location, type, orientation, shape, and texture of crystals. To understand how proteins achieve this exquisite level of control, we are studying the interaction between the phosphoprotein osteopontin (OPN) and the biomineral calcium oxalate monohydrate (COM). In the present study, we have synthesized peptides corresponding to amino acids 220-235 of rat bone OPN (pSHEpSTEQSDAIDpSAEK), one of several highly phosphorylated, aspartic-, and glutamic acid-rich sequences found in the protein. To investigate the role of phosphorylation in interaction with crystals, peptides containing no (P0), one (P1), or all three (P3) phosphates were prepared. Using a novel combination of confocal microscopy and scanning electron microscopy, we show that these peptides adsorb preferentially to {100} faces of COM and inhibit growth of these faces in a phosphorylation-dependent manner. To characterize the mechanism of adsorption of OPN peptides to COM, we have performed the first atomic-scale molecular-dynamics simulation of a protein-crystal interaction. P3 adsorbs to the {100} face much more rapidly than P1, which in turn adsorbs more rapidly than P0. In all cases, aspartic and glutamic acid, not phosphoserine, are the amino acids in closest contact with the crystal surface. These studies have identified a COM face-specific adsorption motif in OPN and delineated separate roles for carboxylate and phosphate groups in inhibition of crystal growth by mineral-associated phosphoproteins. We propose that the formation of close-range, stable, and face-specific interactions is a key factor in the ability of phosphoproteins to regulate biomineralization processes.

Modulation of Calcium Oxalate Dihydrate Growth by Selective Crystal-face Binding of Phosphorylated Osteopontin and Polyaspartate Peptide Showing Occlusion by Sectoral (Compositional) Zoning

“…The formation of these compositional zoning patterns in COD crystals was confirmed by laser scanning confocal microscopy ( Fig. 4c), which enables "optical sectioning" to exclude out-of-focus flare and, thus, allows direct imaging of crystal interiors (20). These two-dimensional zoning patterns vary in appearance with the "depth" of the optical focus and allowing confirmation of the sectoral zoning through different focal planes (Fig.…”

“…Fluorescent molecules and fluorescently-tagged proteins have been previously and effectively used to study protein-mineral interactions (in COM studies (19,20,(77)(78)(79)). Extending this approach to COD and using a poly-Asp 86 -93 peptide not previously examined by others, we have visualized by light and confocal microscopy the incorporation behavior of this peptide into the crystal structure of COD and revealed distinctive and informative three-dimensional sectoral (compositional) zoning patterns (77)(78)(79)(80) that provide significant new insight into protein occlusion into biominerals.…”

Intrinsically Disordered Proteins in Biomineralization

“…Its ubiquitous expression pattern and variations in phosphorylation, glycosylation and sulphation suggest that OPN fulfills many different functions (George & Veis, 2008). Moreover, OPN controls the formation of calcium phosphate (Goldberg & Hunter, 1995), calcium carbonate (Chien et al, 2008), and calcium oxalate (Grohe et al, 2007) crystals. NMR, CD and FTIR studies demonstrated that OPN is an IDP in solution (Tab.1) (Fisher et al, 2001;Gorski et al, 1995).…”

Structural studies of hydrated samples of amorphous calcium phosphate and phosphoprotein nanoclusters

“…Interactions between proteins and crystals, including OPN peptides (Grohe et al 2007), are central to many processes in biomineralisation. The inorganic core of dehydrated CPN samples has however been found to be amorphous, based on experimental results from a range of techniques including electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and X-ray absorption spectroscopy (Cross et al 2005;Holt et al 1982Holt et al , 1996Holt et al and 2009Hunter et al 1996;Holt and Hukins 1991).…”