Understanding the interactions between bone mineral crystals and their binding peptides derived from filamentous phage

Abstract

Hydroxyapatite (HAP) is the main inorganic component of natural bone and assembled along collagen fibers. HAP is formed under the regulation of bone proteins (BPs) including collagen and non-collagenous proteins (NCPs, such as osteocalcin, osteopontin, osteonectin, and bone sialoprotein) during biomineralization. However, the mechanism of the BPs regulating the HAP biomineralization is still unclear and some HAP-interacting domains on BPs have not yet been identified. To understand the interactions between HAP and the peptide motifs, similar to functional motifs on BPs, we employed a sidewall-displayed phage library, instead of a traditional tip-displayed phage library, to discover novel HAP-binding peptides. HAP-binding peptides were expressed as pVIII fusion proteins into the capsid of the filamentous fd phages. By a few rounds of biopanning of the landscape phage library against HAP, we identified multiple HAP-binding peptides. The affinity-selected peptide sequences were aligned to the sequences of BPs using a receptor ligand contacts (RELIC) program, RELIC/MATCH, which is a bioinformatics software that identifies protein-ligand interaction sites and herein discovers the putative HAP-binding domains on these BPs. By both confirming some known HAP-binding domains and identifying new HAP-binding domains, we clarified some mechanisms of HAP-BP interactions. Through a series of binding assays, we discovered that DSSTPSST peptide is the best HAP binder. Using a protein structure prediction model within the Rosetta software suite, we found that the best binding peptides had the lowest peptide-HAP interfacial energy, consistent with the binding assay studies. Simulations suggested that the stable turn-like structure owing to a proline residue in the DSSTPSST peptide enabled favorable electrostatic interactions between aspartate residues in the N-terminal domain and calcium ions in HAP. The phages that displayed DSSTPSST on the sidewall bound and aligned the HAP nanorods along its sidewall, forming HAP-decorated nanofibers. Through a freeze-casting approach, the HAP-decorated nanofibers were assembled into a macroscale scaffold that supported the proliferation of mesenchymal stem cells. Therefore, our work demonstrates a new understanding of HAP-peptide interactions that can be exploited to produce HAP-based biomaterials with potential applications in bone tissue engineering.