Roles of Electrostatics and Conformation in Protein-Crystal Interactions

Abstract

In vitro studies have shown that the phosphoprotein osteopontin (OPN) inhibits the nucleation and growth of hydroxyapatite (HA) and other biominerals. In vivo, OPN is believed to prevent the calcification of soft tissues. However, the nature of the interaction between OPN and HA is not understood. In the computational part of the present study, we used molecular dynamics simulations to predict the adsorption of 19 peptides, each 16 amino acids long and collectively covering the entire sequence of OPN, to the {100} face of HA. This analysis showed that there is an inverse relationship between predicted strength of adsorption and peptide isoelectric point (P<0.0001). Analysis of the OPN sequence by PONDR (Predictor of Naturally Disordered Regions) indicated that OPN sequences predicted to adsorb well to HA are highly disordered. In the experimental part of the study, we synthesized phosphorylated and non-phosphorylated peptides corresponding to OPN sequences 65–80 (pSHDHMDDDDDDDDDGD) and 220–235 (pSHEpSTEQSDAIDpSAEK). In agreement with the PONDR analysis, these were shown by circular dichroism spectroscopy to be largely disordered. A constant-composition/seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC50 = 1.93 µg/ml) and OPN220-235 (IC50 = 1.48 µg/ml) are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC50 = 2.97 µg/ml); the nonphosphorylated version of OPN220-235 has no measurable inhibitory activity. These findings suggest that the adsorption of acidic proteins to Ca2+-rich crystal faces of biominerals is governed by electrostatics and is facilitated by conformational flexibility of the polypeptide chain.

Osteopontin and Wound Healing in Bone

“…Macrophage expression of OPN provides a chemotactic gradient that elicits recruitment of additional macrophages into the wound site that in turn produces additional OPN. Given the mineral-binding properties of OPN in general [Azzopardi et al, 2010], and given our previous report on the ultrastructural localization of OPN in bone wound healing [McKee and Nanci, 1996b], we propose the following scenario for OPN function during the early stages of bone repair after drilling ( fig. 1 ).…”

Gold nanoparticles with different charge and moiety induce differential cell response on mesenchymal stem cell osteogenesis

“…Hence the presence of AuNPs in these intracellular spaces may cause disruption in the internal processes for calcium phosphate deposition in the ECM. In addition, calcium ions are known to preferentially adhere to acidic surfaces [26], hence the presence of many such carboxyl surfaces within the cell could cause dysregulation of the laying of calcium in the mineral matrix. Unlike AuNPeCOOH, the negatively charged AuNPecitrate did not hinder calcium deposition, so the differences between the two treatments might point to a functional group effect.…”

The Flexible Polyelectrolyte Hypothesis of Protein−Biomineral Interaction

“…58 One of the virtual peptides interacting most strongly with HA corresponds to amino acids 65-80 of rat bone OPN (pSHDHMDDDDDDDDDGD). Its adsorption to the {100} face was subsequently studied in detail.…”

“…Shown in Table 1 are the sequences of the 7 virtual peptides found to adsorb best to the {100} face of HA. 58 These sequences are also shown in simplified form in which aspartic acid and glutamic acid are represented as "C", phosphoserine and phosphothreonine as "P", and all other amino acids as "x". According to the strong stereochemical theory, the anionic amino acids should ideally be spaced at every second position along the polypeptide chain in order that all will be on the same side of a β-sheet; according to the weak theory, anionic amino acids should be periodically distributed along the sequence.…”