Calcium carbonate polymorph selection in fish otoliths: A key role of phosphorylation of Starmaker-like protein.

Fish otoliths are calcium carbonate biominerals found in the inner ear commonly used for tracking fish biochronologies and as a model system for biomineralization. The process of fish otolith formation is biologically controlled by numerous biomacromolecules which not only affect crystal size, shape, mechanical properties, but also selection of calcium carbonate polymorph (e.g., aragonite, vaterite). The proteinaceous control over calcium carbonate polymorph selection occurs in many other species (e.g., corals, mollusks, echinoderms) but the exact mechanism of protein interactions with calcium and carbonate ions - constituents of CaCO3 - are not fully elucidated. Herein, we focus on a native Starmaker-like protein isolated from vaterite asteriscus otoliths from Cyprinus carpio. The proteomic studies show the presence of the phosphorylated protein in vaterite otoliths. In a series of in vitro mineralization experiments with Starmaker-like, we show that native phosphorylation is a crucial determinant for the selection of a crystal's polymorphic form. This is the first report showing that the switch in calcium carbonate phase depends on the phosphorylation pattern of a single isolated protein. STATEMENT OF SIGNIFICANCE: Calcium carbonate has numerous applications in industry and medicine. However, we still do not understand the mechanism of biologically driven polymorph selection which results in specific biomineral properties. Previous work on calcium carbonate biominerals showed that either several macromolecular factors or high magnesium concentration (non-physiological) are required for proper polymorph selection (e.g., in mollusk shells, corals and otoliths). In this work, we showed for the first time that protein phosphorylation is a crucial factor for controlling the calcium carbonate crystal phase. This is important because a single protein from the otolith organic matrix could switch between polymorphs depending on the phosphorylation level. It seems that protein post-translational modifications (native, not artificial) are more important for biomolecular control of crystal growth than previously considered.

Effect of Gel Exposition on Calcium and Carbonate Ions Determines the Stm-l Effect on the Crystal Morphology of Calcium Carbonate.

Biomineralization of fish otoliths is regulated by macromolecules, such as proteins, whose presence is crucial for the functionality and properties of these mineralized structures. Special regulatory effects are exerted by intrinsically disordered proteins, such as the polyanionic Starmaker-like protein from medaka, a homolog of zebrafish Starmaker. In this study, we employed a set of bioinspired mineralization experiments with a single diffusion system to investigate the effect of the Starmaker-like protein on calcium carbonate biominerals with regards to the prior exposition of the protein to calcium or carbonate ions. Interestingly, the bioinspired minerals grown in the presence of the Starmaker-like protein in calcium- or carbonate-type experiments differ significantly in terms of morphology and protein distribution within the crystals. Our deeper analysis shows that the Starmaker-like protein action is a result of the environmental conditions to which it is exposed. These findings may be of special interest in the areas of biomineralization process pathways and biomaterial sciences.

Otolin-1, an otolith- and otoconia-related protein, controls calcium carbonate bioinspired mineralization.

BACKGROUND: Otoliths and otoconia are calcium carbonate biomineral structures that form in the inner ear of fish and humans, respectively. The formation of these structures is tightly linked to the formation of an organic matrix framework with otolin-1, a short collagen-like protein from the C1q family as one of its major constituents. METHODS: In this study, we examined the activity of recombinant otolin-1 originating from Danio rerio and Homo sapiens on calcium carbonate bioinspired mineralization with slow-diffusion method and performed crystals characterization with scanning electron microscopy, two-photon excited fluorescence microscopy, confocal laser scanning microscopy and micro-Raman spectroscopy. RESULTS: We show that both proteins are embedded in the core of CaCO3 crystals that form through the slow-diffusion mineralization method. Both of them influence the morphology but do not change the polymorphic mineral phase. D.rerio otolin-1 also closely adheres to the crystal surface. GENERAL SIGNIFICANCE: The results suggest, that otolin-1 is not a passive scaffold, but is directly involved in regulating the morphology of the resulting calcium carbonate biocrystals.

The Role of Intrinsically Disordered Proteins in Liquid-Liquid Phase Separation during Calcium Carbonate Biomineralization.

Some animal organs contain mineralized tissues. These so-called hard tissues are mostly deposits of calcium salts, usually in the form of calcium phosphate or calcium carbonate. Examples of this include fish otoliths and mammalian otoconia, which are found in the inner ear, and they are an essential part of the sensory system that maintains body balance. The composition of ear stones is quite well known, but the role of individual components in the nucleation and growth of these biominerals is enigmatic. It is sure that intrinsically disordered proteins (IDPs) play an important role in this aspect. They have an impact on the shape and size of otoliths. It seems probable that IDPs, with their inherent ability to phase separate, also play a role in nucleation processes. This review discusses the major theories on the mechanisms of biomineral nucleation with a focus on the importance of protein-driven liquid-liquid phase separation (LLPS). It also presents the current understanding of the role of IDPs in the formation of calcium carbonate biominerals and predicts their potential ability to drive LLPS.

Deep blue autofluorescence reflects the oxidation state of human transthyretin.

Human transthyretin (TTR) is a tetrameric protein transporting thyroid hormones and retinol. TTR is a neuroprotective factor and sensor of oxidative stress which stability is diminished due to mutations and aging, leading to amyloid deposition. Adverse environmental conditions, such as redox and metal ion imbalances, induce destabilization of the TTR structure. We have previously shown that the stability of TTR was disturbed by Ca2+ and other factors, including DTT, and led to the formation of an intrinsic fluorophore(s) emitting blue light, termed deep blue autofluorescence (dbAF). Here, we show that the redox state of TTR affects the formation dynamics and properties of dbAF. Free thiols lead to highly unstable subpopulations of TTR and the frequent ocurrence of dbAF. Oxidative conditions counteracted the destabilizing effects of free thiols to some extent. However, strong oxidative conditions led to modifications of TTR, which altered the stability of TTR and resulted in unique dbAF spectra. Riboflavin and/or riboflavin photoproducts bound to TTR and crosslinked TTR subunits. Riboflavin-sensitized photooxidation increased TTR unfolding, while photooxidation, either in the absence or presence of riboflavin, increased proteolysis and resulted in multiple oxidative modifications and dityrosine formation in TTR molecules. Therefore, oxidation can switch the role of TTR from a protective to pathogenic factor.

N'-terminal- and Ca2+-induced stabilization of high-order oligomers of full-length Danio rerio and Homo sapiens otolin-1.

Otolin-1 is a C1q family member and a major component of the organic matrix of fish otoliths and human otoconia. To date, the protein molecular properties have not been characterized. In this work, we describe biochemical characterization and comparative studies on saccular-specific otolin-1 derived from Danio rerio and Homo sapiens. Due to the low abundance of proteins in the otoconial matrix, we developed a production and purification method for both recombinant homologues of otolin-1. Danio rerio and Homo sapiens otolin-1 forms higher-order oligomers that can be partially disrupted under reducing conditions. The presence of Ca2+ stabilizes the oligomers and significantly increases the thermal stability of the proteins. Despite the high sequence coverage, the oligomerization of Danio rerio otolin-1 is more affected by the reducing conditions and presence of Ca2+ than the human homologue. The results show differences in molecular behaviour, which may be reflected in Danio rerio and Homo sapiens otolin-1 role in otolith and otoconia formation.

Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1.

Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca2+. Knowledge of a structure-function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca2+ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca2+, R342W and Q426R were stabilized at higher Ca2+ concentrations than the wild-type form, and R402P was completely insensitive to Ca2+. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).

Molecular mechanism of calcium induced trimerization of C1q-like domain of otolin-1 from human and zebrafish.

The C1q superfamily includes proteins involved in innate immunity, insulin sensitivity, biomineralization and more. Among these proteins is otolin-1, which is a collagen-like protein that forms a scaffold for the biomineralization of inner ear stones in vertebrates. The globular C1q-like domain (gC1q), which is the most conserved part of otolin-1, binds Ca2+ and stabilizes its collagen-like triple helix. The molecular details of the assembly of gC1q otolin-1 trimers are not known. Here, we substituted putative Ca2+-binding acidic residues of gC1q otolin-1 with alanine to analyse how alanine influences the formation of gC1q trimers. We used human and zebrafish gC1q otolin-1 to assess how evolutionary changes affected the function of the protein. Surprisingly, the mutated forms of gC1q otolin-1 trimerized even in the absence of Ca2+, although they were less stable than native proteins saturated with Ca2+. We also found that the zebrafish gC1q domain was less stable than the human homologue under all tested conditions and became stabilized at higher concentrations of Ca2+, which showed that specific interactions leading to the neutralization of the negative charge at the axis of a gC1q trimer by Ca2+ are required for the trimers to form. Moreover, human gC1q otolin-1 seems to be optimized to function at lower concentrations of Ca2+, which is consistent with reported Ca2+ concentrations in the endolymphs of fish and mammals. Our results allow us to explain the molecular mechanism of assembly of proteins from the C1q superfamily, the modulating role of Ca2+ and expand the knowledge of biomineralization of vertebrate inner ear stones: otoliths and otoconia.

Myopathy-Sensitive G-Actin Segment 227-235 Is Involved in Salt-Induced Stabilization of Contacts within the Actin Filament.

Formation of stable actin filaments, critically important for actin functions, is determined by the ionic strength of the solution. However, not much is known about the elements of the actin fold involved in ionic-strength-dependent filament stabilization. In this work, F-actin was destabilized by Cu2+ binding to Cys374, and the effects of solvent conditions on the dynamic properties of F-actin were correlated with the involvement of Segment 227-235 in filament stabilization. The results of our work show that the presence of Mg2+ at the high-affinity cation binding site of Cu-modified actin polymerized with MgCl2 strongly enhances the rate of filament subunit exchange and promotes the filament instability. In the presence of 0.1 M KCl, the filament subunit exchange was 2-3-fold lower than that in the MgCl2-polymerized F-actin. This effect correlates with the reduced accessibility of the D-loop and Segment 227-235 on opposite filament strands, consistent with an ionic-strength-dependent conformational change that modulates involvement of Segment 227-235 in stabilization of the intermonomer interface. KCl may restrict the mobility of the α-helix encompassing part of Segment 227-235 and/or be bound to Asp236 at the boundary of Segment 227-235. These results provide experimental evidence for the involvement of Segment 227-235 in salt-induced stabilization of contacts within the actin filament and suggest that they can be weakened by mutations characteristic of actin-associated myopathies.

Functional derivatives of human dentin matrix protein 1 modulate morphology of calcium carbonate crystals.

Dentin matrix protein 1 (DMP1) is an acidic, extracellular matrix protein essential for biomineralization of calcium phosphate, in bone and dentin. It is proteolytically processed into two fragments, 44K and 56K. Recently, the presence of DMP1 was noticed in inner ear, specifically in otoconia, which are calcium carbonate biominerals involved in sensing of balance. In this study, the solution structure and biomineralization activity of otoconial 44K and 56K fragments toward calcium carbonate were investigated. The results of analytical ultracentrifugation, circular dichroism, and gel filtration indicated that DMP1 fragments are disordered in solution. Notably, 56K formed oligomers in the presence of calcium ions. It was also observed that both fragments influenced the crystal growth by in vitro biomineralization assay and scanning electron microscopy. In addition, they sequester the calcium ions during the calcite formation. Calcium carbonate crystals precipitated in vitro changed their size and shape in the presence of DMP1 fragments. Oligomerization propensity of 56K may significantly enhance this function. Our study indicates that intrinsically disordered DMP1 has a previously unknown regulatory function for biomineralization of otoconia.

In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Otoliths are one of the biominerals whose formation is highly controlled by proteins. The first protein discovered to be involved in otolith biomineralization in zebrafish was starmaker (Stm). Previously, Stm was shown to be responsible for the preferential formation of aragonite, a polymorph of calcium carbonate, in otoliths. In this work, proteomic analysis of adult zebrafish otoliths was performed. Stm is the only highly phosphorylated protein found in our studies. Besides previously studied otolith proteins, we discovered several dozens of unknown proteins that reveal the likely mechanism of biomineralization. A comparison of aragonite and vaterite otoliths showed similarities in protein composition. We observed the presence of Stm in both types of otoliths. In vitro studies of 2 characteristic Stm fragments indicated that the DS-rich region has a special biomineralization activity, especially after phosphorylation.-Kalka, M., Markiewicz, N., Ptak, M., Sone, E. D., Ozyhar, A., Dobryszycki, P., Wojtas, M. In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Effect of calcium ions on structure and stability of the C1q-like domain of otolin-1 from human and zebrafish.

Otolin-1 is a collagen-like protein expressed in the inner ear of vertebrates. It provides an organic scaffold for otoliths in fish and otoconia in land vertebrates. In this study, the expression and purification procedure of C1q-like domain of otolin-1 from human and zebrafish was developed. The structure and stability of the proteins were investigated. The results of sedimentation velocity analytical ultracentrifugation and small-angle X-ray scattering indicated that the C1q-like domain of otolin-1 forms stable trimers in solution in the presence of calcium ions. It was also observed that calcium ions influenced the secondary structure of the proteins. C1q-like domains were stabilized by the calcium ions. The human variant was especially affected by the calcium ions. The results indicate the importance of the C1q-like domain for the assembly of the organic matrix of otoliths and otoconia.

Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64).

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed.

Dimeric and tetrameric forms of muscle fructose-1,6-bisphosphatase play different roles in the cell.

Muscle fructose 1,6-bisphosphatase (FBP2), besides being a regulatory enzyme of glyconeogenesis also protects mitochondria against calcium stress and plays a key role in regulation of the cell cycle, promoting cardiomyocytes survival. However, in cancer cells, FBP2 acts as an anti-oncogenic/anti-proliferative protein. Here, we show that the physiological function of FBP2 depends both on its level of expression in a cell as well as its oligomerization state. Animal fructose-1,6-bisphosphatases are thought to function as tetramers. We present evidence that FBP2 exists in an equilibrium between tetramers and dimers. The dimeric form is fully active and insensitive to AMP, the main allosteric inhibitor of FBP2. Tetramerization induces the sensitivity of the protein to AMP, but it requires the presence of a hydrophobic central region in which leucine 190 plays a crucial role. Only the tetrameric form of FBP2 is retained in cardiomyocyte cell nucleus whereas only the dimeric form associates with mitochondria and protects them against stress stimuli, such as elevated calcium and H2O2 level. Remarkably, in hypoxic conditions, which are typical for many cancers, FBP2 ceases to interact with mitochondria and loses its pro-survival potential. Our results throw new light on the basis of the diverse role of FBP2 in cells.

Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 µM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 µM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails.

Two major lipophilic hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64) and Tribolium castaneum (TcChd64). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH) domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV) circular dichroism (CD) spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC) revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG)-like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners.

[Biomineralization--precision of shape, structure and properties controlled by proteins]. / Biomineralizacja--kontrolowana przez bialka precyzja ksztaltu, struktury i wlasciwosci.

ABSTRACT Biomineralization is the process of the formation of crystal structures that is under biological control. Living organisms produce structures such as bone, teeth, otoliths, otoconia or shells. Although the chemical composition of these tissues is similar to corresponding inorganic minerals, their structure and mechanical properties differ significantly. This may be because of how they are adapted for the functions they perform. The precise control of the formation of biominerals starting with the early nucleation stage influences how the final tissues are formed. The key factors which determine the size, shape, internal structure and properties of biominerals are proteins which control the nucleation and growth of the crystals. Biomineralization is a multi-step process involving protein-protein interactions, as well as interactions between proteins and inorganic fraction. Due to their specific properties, intrinsically disordered proteins (IDPs) perform a particularly important role in the control of the biomineralization process. This article contains an overview of biominerals that are naturally occurring and describes the structures and mineralization mechanisms of the most important of them. The main part of this work was dedicated to the role of proteins which control crystal growth.

The molecular basis of conformational instability of the ecdysone receptor DNA binding domain studied by in silico and in vitro experiments.

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of our study was an elucidation of the molecular basis of this intriguing instability. Toward this end, we mapped the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the α-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. Our results might serve as a benchmark for further studies of the intricate nature of the EcR molecule.

The effect of counter ions on the conformation of intrinsically disordered proteins studied by size-exclusion chromatography.

Counter ions are able to change the conformation of intrinsically disordered proteins (IDPs) to a more compact structure via the reduction of electrostatic repulsion. When the extended IDP conformation is transformed into a more ordered one, the value of the Stokes radius should decrease. Size-exclusion chromatography is a simple method for the determination of the Stokes radius, which describes the hydrodynamic properties of protein molecules. In our paper size-exclusion chromatography experiments of Starmaker (a highly acidic IDP), in the presence of various counter ions, are presented as an example of a simple experimental method, which provides valuable information about subtle counter ions-induced conformational changes in IDP.

Conformational changes in the DNA-binding domains of the ecdysteroid receptor during the formation of a complex with the hsp27 response element.

The ecdysone receptor (EcR) and the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids that initiates metamorphosis in insects. The Usp and EcR DNA-binding domains (UspDBD and EcRDBD, respectively) form a heterodimer on the natural pseudopalindromic element from the hsp27 gene promoter. The conformational changes in the protein-DNA during the formation of the UspDBD-EcRDBD-hsp27 complex were analyzed. Recombined UspDBD and EcRDBD proteins were purified and fluorescein labeled (FL) using the intein method at the C-ends of both proteins. The changes in the distances from the respective C-ends of EcRDBD and/or UspDBD to the 5'- and/or 3'-end of the response element were measured using fluorescence resonance energy transfer (FRET) methodology. The binding of EcRDBD induced a strong conformational change in UspDBD and caused the C-terminal fragment of the UspDBD molecule to move away from both ends of the regulatory element. UspDBD also induced a significant conformational change in the EcRDBD molecule. The EcRDBD C-terminus moved away from the 5'-end of the regulatory element and moved close to the 3'-end. An analysis was also done on the effect that DHR38DBD, the Drosophila ortholog of the mammalian NGFI-B, had on the interaction of UspDBD and EcRDBD with hsp27. FRET analysis demonstrated that hsp27 bending was induced by DHR38DBD. Fluorescence data revealed that hsp27 had a shorter end-to-end distance both in the presence of EcRDBD as well as in the presence of EcRDBD together with DHR38DBD, with DNA bend angles of about 36.2° and 33.6°, respectively. A model of how DHR38DBD binds to hsp27 in the presence of EcRDBD is presented.