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.

Molecular differences in collagen organization and in organic-inorganic interfacial structure of bones with and without osteocytes.

Bone is a fascinating biomaterial composed mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, and water molecules residing at the interfaces between these phases. They are hierarchically organized with minor constituents such as non-collagenous proteins, citrate ions and glycosaminoglycans into a composite structure that is mechanically durable yet contains enough porosity to accommodate cells and blood vessels. The nanometer scale organization of the collagen fibrous structure and the mineral constituents in bone were recently extensively scrutinized. However, molecular details at the lowest hierarchical level still need to be unraveled to better understand the exact atomic-level arrangement of all these important components in the context of the integral structure of the bone. In this report, we unfold some of the molecular characteristics differentiating between two load-bearing (cleithrum) bones, one from sturgeon fish, where the matrix contains osteocytes and one from pike fish where the bone tissue is devoid of these bone cells. Using enhanced solid-state NMR measurements, we underpin disparities in the collagen fibril structure and dynamics, the mineral phases, the citrate content at the organic-inorganic interface and water penetrability in the two bones. These findings suggest that different strategies are undertaken in the erection of the mineral-organic interfaces in various bones characterized by dissimilar osteogenesis or remodeling pathways and may have implications for the mechanical properties of the particular bone. STATEMENT OF SIGNIFICANCE: Bone boasts unique interactions between collagen fibers and mineral phases through interfaces holding together this bio-composite structure. Over evolution, fish have gone from mineralizing their bones aided by certain bone cells called osteocytes, like tetrapod, to mineralization without these cells. Here, we report atomic level differences in collagen fiber cross linking and organization, porosity of the mineral phases and content of citrate molecules at the bio-mineral interface in bones from modern versus ancient fish. The dissimilar structural features may suggest disparate mechanical properties for the two bones. Fundamental level understanding of the organic and inorganic components in bone and the interfacial interactions holding them together is essential for successful bone repair and for treating better tissue pathologies.

Structure and Dynamics Perturbations in Ubiquitin Adsorbed or Entrapped in Silica Materials Are Related to Disparate Surface Chemistries Resolved by Solid-State NMR Spectroscopy.

Protein immobilization on material surfaces is emerging as a powerful tool in the design of devices and active materials for biomedical and pharmaceutical applications as well as for catalysis. Preservation of the protein's biological functionality is crucial to the design process and is dependent on the ability to maintain its structural and dynamical integrity while removed from the natural surroundings. The scientific techniques to validate the structure of immobilized proteins are scarce and usually provide limited information as a result of poor resolution. In this work, we benchmarked the ability of standard solid-state NMR techniques to resolve the effects of binding to dissimilar silica materials on a model protein. In particular, the interactions between ubiquitin and the surfaces of MCM41, SBA15, and silica formed in situ were tested for their influence on the structure and dynamics of the protein. It is shown that the protein's globular fold in the free state is only slightly perturbed in the three silica materials. Local motions on a residue level that are quenched by immobilization or, conversely, that arise from the process are also detailed. NMR measurements show that these perturbations are unique to each silica material and can serve as reporters of the characteristic surface chemistry.

Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites.

Details of apatite formation and development in bone below the nanometer scale remain enigmatic. Regulation of mineralization was shown to be governed by the activity of non-collagenous proteins with many bone diseases stemming from improper activity of these proteins. Apatite crystal growth inhibition or enhancement is thought to involve direct interaction of these proteins with exposed faces of apatite crystals. However, experimental evidence of the molecular binding events that occur and that allow these proteins to exert their functions are lacking. Moreover, recent high-resolution measurements of apatite crystallites in bone have shown that individual crystallites are covered by a persistent layer of amorphous calcium phosphate. It is therefore unclear whether non-collagenous proteins can interact with the faces of the mineral crystallites directly and what are the consequences of the presence of a disordered mineral layer to their functionality. In this work, the regulatory effect of recombinant osteopontin on biomimetic apatite is shown to produce platelet-shaped apatite crystallites with disordered layers coating them. The protein is also shown to regulate the content and properties of the disordered mineral phase (and sublayers within it). Through solid-state NMR atomic carbon-phosphorous distance measurements, the protein is shown to be located in the disordered phases, reaching out to interact with the surfaces of the crystals only through very few sidechains. These observations suggest that non-phosphorylated osteopontin acts as regulator of the coating mineral layers and exerts its effect on apatite crystal growth processes mostly from afar with a limited number of contact points with the crystal.

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.

How does osteocalcin lacking γ-glutamic groups affect biomimetic apatite formation and what can we say about its structure in mineral-bound form?

Non-collagenous proteins such as osteocalcin function as regulators of the mineralization process in bone. Osteocalcin undergoes post-translational modification adding an extra carboxylate group on three of its glutamate residues to enhance interaction with bone mineral. In this work, we examine regulation of biomimetic apatite formation by osteocalcin that was not modified after translation. We analyze the structural features in the protein and mineral-protein interfaces to elicit the unmodified protein's fold inside the mineral and to unveil the species that interact with the mineral surface. The results presented here give clues on the protein's active role in controlling the mineral phases that are formed on hydroxyapatite crystals and its ability to influence the extent of order in these crystals.

Dynamics in hydrophilic and hydrophobic molecular chains tethered to MCM41-type mesoporous silica upon wetting and dehydration processes.

Surface modified mesoporous silica materials are important materials for heterogeneous catalysis and are attracting attention as potential drug carriers. The functionality of these materials relies on the physical and chemical properties of the tethers attached to MCM41 silica surface. These chemically linked tails act as molecular brushes, that can capture pollutant molecules, anchor points for catalysts and can host drug molecules. To utilize the full potential of the tailored silica surfaces, one should infer their properties at different levels of solvation. Here, 1H MAS NMR spectroscopy is used to monitor the dynamic properties of two modified MCM41 materials, an aminopropyl tethered MCM41 and an octyl tethered MCM41, through the process of controlled hydration. The surface site resolved measurements demonstrate how the chemical nature of the two tethers governs the way water molecules are directed to the different sites in the porous materials.

Ubiquitin immobilized on mesoporous MCM41 silica surfaces - Analysis by solid-state NMR with biophysical and surface characterization.

Deriving the conformation of adsorbed proteins is important in the assessment of their functional activity when immobilized. This has particularly important bearings on the design of contemporary and new encapsulated enzyme-based drugs, biosensors, and other bioanalytical devices. Solid-state nuclear magnetic resonance (NMR) measurements can expand our molecular view of proteins in this state and of the molecular interactions governing protein immobilization on popular biocompatible surfaces such as silica. Here, the authors study the immobilization of ubiquitin on the mesoporous silica MCM41 by NMR and other techniques. Protein molecules are shown to bind efficiently at pH 5 through electrostatic interactions to individual MCM41 particles, causing their agglutination. The strong attraction of ubiquitin to MCM41 surface is given molecular context through evidence of proximity of basic, carbonyl and polar groups on the protein to groups on the silica surface using NMR measurements. The immobilized protein exhibits broad peaks in two-dimensional 13C dipolar-assisted rotational resonance spectra, an indication of structural multiplicity. At the same time, cross-peaks related to Tyr and Phe sidechains are missing due to motional averaging. Overall, the favorable adsorption of ubiquitin to MCM41 is accompanied by conformational heterogeneity and by a major loss of motional degrees of freedom as inferred from the marked entropy decrease. Nevertheless, local motions of the aromatic rings are retained in the immobilized state.

The Uptake and Assembly of Alkanes within a Porous Nanocapsule in Water: New Information about Hydrophobic Confinement.

In Nature, enzymes provide hydrophobic cavities and channels for sequestering small alkanes or long-chain alkyl groups from water. Similarly, the porous metal oxide capsule [{Mo(VI) 6 O21 (H2 O)6 }12 {(Mo(V) 2 O4 )30 (L)29 (H2 O)2 }](41-) (L=propionate ligand) features distinct domains for sequestering differently sized alkanes (as in Nature) as well as internal dimensions suitable for multi-alkane clustering. The ethyl tails of the 29 endohedrally coordinated ligands, L, form a spherical, hydrophobic "shell", while their methyl end groups generate a hydrophobic cavity with a diameter of 11 Šat the center of the capsule. As such, C7 to C3 straight-chain alkanes are tightly intercalated between the ethyl tails, giving assemblies containing 90 to 110 methyl and methylene units, whereas two or three ethane molecules reside in the central cavity of the capsule, where they are free to rotate rapidly, a phenomenon never before observed for the uptake of alkanes from water by molecular cages or containers.

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.

NMR studies of DNA microcapsules prepared using sonochemical methods.

DNA molecules were recently converted using ultrasonic irradiation into microcapsules that can trap hydrophobic molecules in aqueous solution. These DNA microcapsules are capable of penetrating prokaryotic and eukaryotic cells, delivering drugs and transferring genetic information e.g. for protein expression into the host cells. DNA molecules of different sizes and structures can be assembled into spherical capsules, but to date, the interactions that hold them together in these large structural constructs are unknown. In the current study, capsules prepared from a 12 base double helix DNA were investigated using NMR spectroscopy. Solution NMR studies of the DNA emulsion reveal DNA molecules with a perturbed structure with a size similar to the precursor DNA based on diffusion NMR measurements. 2D NMR correlation measurements and chemical shift perturbation analysis show partial unzipping of AT base pairs in the centre of the modified duplex, freeing nucleoside bases to interact with other bases on other precursor molecules thereby facilitating aggregation. Slow tumbling of the microspheres renders them invisible in solution NMR spectra; therefore magic angle spinning NMR measurements are performed which provide limited evidence of the DNA in the microcapsule state.

Dynamic behavior of supramolecular comb polymers consisting of poly(2-vinyl pyridine) and palladium-pincer surfactants in the solid state.

When poly(2-vinyl pyridine) is combined with Pd-pincer-based organometallic surfactants, a mesomorphic structure forms due to weak stacking interactions between the pyridine units and the Pd-pincer headgroups. The weak binding between the surfactant and the polymer competes with the tendency of the aliphatic tails of the surfactant to crystallize. Here, we demonstrate that over extended periods of incubation, the crystallization tendency of the surfactant tails causes the surfactant molecules to detach from the polymer and gives rise to additional packing modes of the alkyl tails featuring higher crystalline order. The dynamic behavior of these aged structures was investigated by variable-temperature small-angle X-ray scattering (SAXS) and solid-state (13)C NMR, and revealed the influence of thermal changes on the molecular level, and how these changes propagate to the mesoscale structure.

Elucidating the role of stable carbon radicals in the low temperature oxidation of coals by coupled EPR-NMR spectroscopy - a method to characterize surfaces of porous carbon materials.

Recently, the nature of the carbon radicals stabilized in various coals was characterized using Electron Paramagnetic Resonance (EPR) spectroscopy. It was demonstrated that introducing diamagnetic gases, such as He, CO2, or N2, under STP conditions to the coal surface induces the appearance of a new type of carbon surface radical. This interesting phenomenon was not observed for all coal types, which suggests that the use of EPR measurements can provide information on functional groups that exist on the carbon surface. In the current study coupling Nuclear Magnetic Resonance (NMR) with gas flow in situ EPR measurements significantly enhances the ability to characterize the nature of these radicals and the surface functional groups of coal samples. It was observed that the oxidative reaction with aliphatic groups leads to the increase in stable carbon centered radicals. In addition, there are some species of carbon centered radicals that show reversible binding to O2. This phenomena, however, is dependent on the coal rank, sample porosity and the degree of the coal sample to undergo structural changes under the LTO process. These findings shed new light onto the complex heterogeneous low temperature oxidation reactions occurring at the coal surface.

1,1-diamino-2,2-dinitroethylenes are always zwitterions.

The nitration of tetraiodoethylene (7) yields 1,1-diiodo-2,2-dinitroethylene (8). The latter reacts with alkylamines 9 or alkyldiamines 11 to give the corresponding acyclic 1,1-diamino-2,2-dinitroethylenes 10 or their cyclic analogs 12, respectively. On the basis of liquid and solid-state (13)C and (15)N NMR data, x-ray analysis and ab initio calculations, we suggest that the title compounds are always zwitterionic and that the C(A)-C(N) bond is not a true double bond.

23Na and 2H magnetic resonance studies of osteoarthritic and osteoporotic articular cartilage.

In this study, the short component of the (23)Na T(2) (T(2f)) and the (23)Na and (2)H quadrupolar interactions (nu(Q)) were measured in bone-cartilage samples of osteoarthritic (OA) and osteoporotic (OP) patients. (23)Na nu(Q) was found to increase in osteoarthritic articular cartilage relative to controls. Similar results were found in bovine cartilage following proteoglycan (PG) depletion, a condition that prevails in osteoarthritis. (23)Na nu(Q) and 1/T(2f) for articular cartilage obtained from osteoporotic patients were significantly larger than for control and osteoarthritic cartilage. Decalcification of both human and bovine articular cartilage resulted in an increase of (23)Na nu(Q) and 1/T(2f), showing the same trend as the osteoporotic samples. Differences in the ratio of the intensity of the large (2)H splitting to that of the small one in the calcified zone were also observed. In osteoporosis, this ratio was twice as large as that obtained for both control and osteoarthritic samples. The (2)H and (23)Na results can be interpreted as due to sodium ions and water molecules filling the void created by the calcium depletion and to calcium ions being located in close association with the collagen fibers. To the best of our knowledge, this is the first study reporting differences of NMR parameters in cartilage of osteoporotic patients.

Monitoring of the effect of intervertebral disc nucleus pulposus ablation by MRI.

In order to investigate intervertebral disc (IVD) degeneration and repair, a quantitative non-invasive tool is needed. Various MRI methods including qCPMG, which yields dipolar echo relaxation time (T(DE)), magnetization transfer contrast (MTC), and (1)H and (2)H double quantum filtered (DQF) MRI were used in the present work to monitor changes in rat IVD after ablation of the nucleus pulposus (NP), serving as a model of severe IVD degeneration. In the intact IVD, a clear distinction between the annulus fibrosus (AF) and the NP is obtained on T(2) and T(DE) weighted images as well as on MTC maps, reflecting the high concentration of ordered collagen fibers in the AF. After ablation of the NP, the distinction between the compartments is lost. T(2) and T(DE) relaxation times are short throughout the disc and MTC is high. (1)H and (2)H DQF signal, which in intact discs is obtained only for the AF, is now observable throughout the tissue. These results indicate that after ablation, there is an ingression of collagen fibers from the AF into the area that was previously occupied by the NP, as was confirmed by histology.

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.

Multinuclear NMR and MRI studies of the maturation of pig articular cartilage.

The maturation of pig articular cartilage was followed by (2)H in-phase double quantum filtered (IP-DQF) spectroscopic MRI, (1)H T(2) MRI, and (23)Na DQF and triple quantum filtered MRS. The results all lead to the conclusion that the order and density of the collagen fibers in articular cartilage increase from birth to maturity. At birth, both (2)H IP-DQF signal and (1)H T(2) were homogeneous throughout the cartilage and their values independent of the orientation of the plug relative to the magnetic field. At maturation, the (2)H IP-DQF spectrum near the bone is composed of two pairs of quadrupolar split satellites and the (1)H T(2) relaxation is biexponential, indicating the presence of two groups of collagen fibers. The (2)H satellites are orientation dependent, indicating that the two groups of fibers are well ordered at maturation. The fast component of (1)H T(2) is also orientation dependent and thus we have concluded that this component results from residual dipolar interaction, while the slow T(2) component in mature cartilage, as well as the T(2) relaxation in immature cartilage, is governed by other mechanisms.

The effect of decalcification on the microstructure of articular cartilage assessed by 2H double quantum filtered spectroscopic MRI.

2H quadrupolar splitting of deuterated water molecules is a sensitive measure of the order and density of the collagen fibers in articular cartilage. In the calcified zone, near the bone, two pairs of quadrupolar split satellites were previously observed. To examine whether the large splitting observed originates from the presence of calcium ions and hydroxyapatite, one-dimensional 2H single and double quantum filtered spectroscopic imaging were performed on articular cartilage-bone plugs before and after decalcification. After decalcification, the magnitude splitting of the two pairs of satellites did not change and orientation dependency was kept. However, the intensity of the large splitting was greatly enhanced. According to these results the two pairs of satellites do not stem from the presence of calcium ions and hydroxyapatite but originate from the presence of two groups of collagen fibers with different degrees of hydration. The enhanced intensity of the large splitting is attributed to an increased amount of water molecules that fill the void, resulting from the removal of hydroxyapatite, which resides near the fibers responsible for the large splitting. The quadrupolar splitting observed in the trabecular bone was not orientation-dependent, indicating a random orientation of the collagen fibers in that tissue.

The effect of detachment of the articular cartilage from its calcified zone on the cartilage microstructure, assessed by 2H-spectroscopic double quantum filtered MRI.

Most studies on articular cartilage properties have been conducted after detachment of the cartilage from the bone. In the present work we investigated the effect of detachment on collagen fiber architecture. We used one-dimensional (2)H double quantum filtered MRI on cartilage bone plugs equilibrated in deuterated saline. The quadrupolar splittings observed in the different zones were related to the degree of order and the density of the collagen fibers. The method is non-destructive, allowing for measurements on the same plug without the need for fixation, dehydration, sectioning and decalcification. Detachment of the radial from the calcified zone resulted in swelling of the cartilage plug in physiological saline and a concomitant decrease in the quadrupolar splitting. The effect of mechanical pressure on the (2)H quadrupolar splittings for the detached cartilage and for the calcified zone-bone plugs were compared with those of the same zones in the intact cartilage-bone plug. The splitting in the radial zone of the detached cartilage collapsed at much smaller loads compared to the intact cartilage-bone plug. The effect of the load on the size of the cartilage was also greater for the detached plug. These results indicate that anchoring of the cartilage to the bone through the calcified zone plays an important role in retaining the order of the collagen fibers. The water (2)H quadrupolar splitting in intact and proteoglycan-depleted cartilage was the same, indicating that the proteoglycans do not contribute to the ordering of the collagen fibers.