Structural insights into the role of the WW2 domain on tandem WW-PPxY motif interactions of oxidoreductase WWOX.

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW-peptide interactions is not always intuitive. The WW domain-containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1-WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.

Peptides from diatoms and grasses harness phosphate ion binding to silica to help regulate biomaterial structure.

Many life forms generate intricate submicron biosilica structures with various important biological functions. The formation of such structures, from the silicic acid in the waters and in the soil, is thought to be regulated by unique proteins with high repeats of specific amino acids and unusual sidechain modifications. Some silicifying proteins are characterized by high prevalence of basic amino acids in their primary structures. Lysine-rich domains are found, for instance, in diatom silaffin proteins and in the sorghum grass siliplant1 protein. These domains exhibit catalytic activity in silica chain condensation, owing to molecular interactions of the lysine amine groups with the forming mineral. The use of amine chemistry by two very remote organisms has motivated us to seek other molecular biosilicification processes that may be common to the two life forms. In diatom silaffins, domains rich in phosphoserine residues are thought to assist the assembly of silaffin molecules into an organic supra-structure which serves as a template for the silica to precipitate on. This mold, held by salt bridges between serine phosphates and lysine amines, dictates the shape of the silica particles formed. Yet, silica synthesized with the dephosphorylated silaffin in phosphate buffer showed similar morphology to the one prepared with the native protein, suggesting that a defined spatial arrangement of serine phosphates is not required to generate silica with the desired shape. Concurrently, free phosphates enhanced the activity of siliplant1 in silica formation. It is therefore beneficial to characterize the involvement of these anions as co-factors in regulated silicification by functional peptides from the two proteins and to understand whether they play similar molecular role in the mechanism of mineralization. Here we analyze the molecular interactions of free phosphate ions with silica and the silaffin peptide PL12 and separately with silica and siliplant1 peptide SLP1 in the two biomimetic silica products generated by the two peptides. MAS NMR measurements show that the phosphate ions interact with the peptides and at the same time may be forming bonds with the silica mineral. This bridging capability may add another avenue by which the structure of the silica material is influenced. A model for the molecular/ionic interactions at the bio-inorganic interface is described, which may have bearings for the role of phosphorylated residues beyond the function as intermolecular cross linkers or free phosphate ions as co-factors in regulation of silicification. STATEMENT OF SIGNIFICANCE: The manuscript addresses the question how proteins in diatoms and plants regulate the biosilica materials that are produced for various purposes in organisms. It uses preparation of silica in vitro with functional peptide derivatives from a sorghum grass protein and from a diatom silaffin protein separately to show that phosphate ions are important for the control that is achieved by these proteins on the final shape of the silica material produced. It portrays via magnetic resonance spectroscopic measurements, in atomic detail, the interface between atoms in the peptide, atoms on the surface of the silica formed and the phosphate ions that form chemical bonds with atoms on the silica as part of the mechanism of action of these peptides.

Changes to the Disordered Phase and Apatite Crystallite Morphology during Mineralization by an Acidic Mineral Binding Peptide from Osteonectin.

Noncollagenous proteins regulate the formation of the mineral constituent in hard tissue. The mineral formed contains apatite crystals coated by a functional disordered calcium phosphate phase. Although the crystalline phase of bone mineral was extensively investigated, little is known about the disordered layer's composition and structure, and less is known regarding the function of noncollagenous proteins in the context of this layer. In the current study, apatite was prepared with an acidic peptide (ON29) derived from the bone/dentin protein osteonectin. The mineral formed comprises needle-shaped hydroxyapatite crystals like in dentin and a stable disordered phase coating the apatitic crystals as shown using X-ray diffraction, transmission electron microscopy, and solid-state NMR techniques. The peptide, embedded between the mineral particles, reduces the overall phosphate content in the mineral formed as inferred from inductively coupled plasma and elemental analysis results. Magnetization transfers between disordered phase species and apatitic phase species are observed for the first time using 2D (1)H-(31)P heteronuclear correlation NMR measurements. The dynamics of phosphate magnetization transfers reveal that ON29 decreases significantly the amount of water molecules in the disordered phase and increases slightly their content at the ordered-disordered interface. The peptide decreases hydroxyl to disordered phosphate transfers within the surface layer but does not influence transfer within the bulk crystalline mineral. Overall, these results indicate that control of crystallite morphology and properties of the inorganic component in hard tissue by biomolecules is more involved than just direct interaction between protein functional groups and mineral crystal faces. Subtler mechanisms such as modulation of the disordered phase composition and structural changes at the ordered-disordered interface may be involved.

Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells.

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.