The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide—Localization of a putative binding site

Abstract

Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO )-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO -binding capabilities with TiBP. Intrigued by the unique TiO -binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO , indicating that neither the enzyme undergoes major conformational changes nor the TiO binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO through similar regions located far from the active site and the dimerization sites. The putative TiO -binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins.

RGD‐modified dihydrolipoamide dehydrogenase as a molecular bridge for enhancing the adhesion of bone forming cells to titanium dioxide implant surfaces

“…Conversely, DLDH forms an exceptionally strong interaction with TiO 2. This interaction is based on a specific binding site and involves coordinative bonding via CHED residues …”

“…Previously, it was calculated that about 2000 protein molecules (equivalent to 8000 RGD moieties) were bound per μm 2 . The strong binding of DLDH to TiO 2 was shown to be dissociated only in the presence of high molar concentrations of urea …”

“…Another approach to achieve more effective osseointegration is based on boosting the integrin‐RGD interaction between the cells and the metal oxide layer. Osteoblasts express a variety of extracellular markers, including integrins, which allow binding with surrounding proteins containing RGD recognition sites . A chimeric peptide consisting of an RGD sequence and RKLPDA was used to mediate MC3T3‐E1 cell adhesion .…”

“…Unfortunately, many of them interact with TiO 2 by weak associations consisting of ionic forces and hydrogen bonds and resulting in an unstable, highly reversible protein adsorption . In contrast, the binding of the mitochondrial enzyme dihydrolipoamide dehydrogenase (DLDH) to the TiO 2 surface which has been shown to be based on specific coordinative bonds involving CHED motifs, present on specific recognition sites on the protein molecule creates much stronger bonding …”

“…The interaction between osteoblasts and the implant's surface is the first step that provides better functionality in the osseointegration process. Osteoblasts express a variety of membrane proteins including integrins that mediates their binding via RGD‐specific recognition sites . Bone marrow stromal cells, MBA‐15 were extensively studied for their bone‐forming characteristics, both in vitro and in vivo.…”