Original Full Length ArticleStructural disorder in proteins brings order to crystal growth in biomineralization
Highlights
► Proteins, annotated for biomineralization, show an extremely high level of predicted disorder. ► Precipitation and maturation in biomineralization is strictly related to highly disordered proteins. ► The process is evolutionary linked to disordered proteins in all known mineralization types. ► Biased residue composition is only compatible with protein disorder.
Introduction
It has become generally accepted lately that many proteins or regions of proteins (intrinsically disordered proteins/regions, IDPs/IDRs) lack a well-defined three-dimensional structure under native conditions [1], [2], [3], [4]. These proteins are enriched in charged/polar amino acids (e.g. Gln, Ser, Pro, Glu, Lys, Gly) and are depleted in hydrophobic residues (e.g. Tyr, Trp, Phe, Leu, Ile), which are responsible for their structure adopting multiple, rapidly interconverting conformational states. The frequency of structural disorder is particularly high in regulatory and signaling proteins, such as transmembrane receptors [5], intracellular scaffold proteins [6], [7], chaperones [8] and transcription factors [9]. Because of its link with regulatory functions, structural disorder of proteins is also frequently observed in disease-associated proteins, such as p53, BRCA1, α-synuclein or tau [10]. Biophysical evidence is available for the structural disorder of more than 1300 proteins/regions [11], whereas bioinformatic predictions suggest that in higher eukaryotes more than 50% of proteins are likely to contain long disordered regions [12]. The high frequency of structural disorder that stems from the functional advantages disorder confers on proteins, such as increased speed of interactions [13], [14], uncoupling of specificity from binding strength [2], the capacity to interact with multiple interacting partners [15], [16] and the ability to carry out several functions, also termed gene sharing or moonlighting [17].
Partial or full structural disorder has also been reported in some proteins involved in biomineralization [18], [19], the biological process which results in the formation of hard tissues (bone, dentin, enamel, eggshell, seashells, marine diatom silica walls, etc.) in living organisms. In a molecular sense, biomineralization embodies the processes in which living organisms accumulate and deposit crystalline inorganic material in their body as an outer frame for protection (e.g. oyster shell), an inner frame for strength and locomotion (e.g. vertebrate bone), or comminuting organs for predation and feeding (e.g. teeth and gastroliths). In all cases, controlled mineralization starts with the production of an organic frame within or upon which orderly crystallization can take place [20]. In dentin and bone this is followed by the secretion of doubly interactive proteins which bind to this assembled framework and nucleate mineral formation, however in the case of enamel, amelogenins serve as both extracellular matrix scaffold and hydroxyapatite (HAP) interacting agent [21], [22].
A subset of these interactive molecules and other proteins are involved in inhibition and regulation thus they control the direction and extent of crystal growth [23]. Often, the proteins involved lack identifiable ordered domains and are reported to be structurally disordered. For example, dentin sialophosphoprotein (DSPP), one of the main regulators of nucleation in dentin mineralization, is 1301 residues in length and is processed into two major segments: DSP and DPP, of which DPP is confirmed to have disordered structure by NMR [24]. Osteopontin (OPN), one of the main regulators of crystallization in both bone and teeth [25], [26], [27], [28], [29] and bone sialoprotein-2 (BSP), which in vitro initiates the HAP crystal formation [26], [30] and has effect on bone remodeling in vivo [31], [32], [33], have also been reported to have random structure by NMR. Dentin matrix protein-1 (DMP) which promotes HAP formation when bound to collagen and inhibits crystal formation when free in solution, also shows random-coil structure by circular dichroism (CD) and IR [34], [35], [36], [37], [38]. The secondary structure of some key proteins in biomineralization was sporadically characterized by various biophysical techniques (CD, infrared spectroscopy, AFM, dynamic light scattering) [28], [36], [39], [40], [41]. Caseins of closely related function, which inhibit the deposition of HAP in biological fluids rich in calcium such as blood and milk, have also been reported to be largely disordered [42]. While the binding of disordered proteins is often coupled with folding resulting in well structured (secondary structure) region(s) in the complex, the proteins involved in biomineralization were shown to bind as flexible polyelectrolytes and to bind to HAP with minimal changes in secondary structure [36], suggesting the importance of the disordered state [43].
Although these observations imply the role of structural disorder of proteins in biomineralization, the generality of this correlation is uncertain, because the experimental approaches applied only provide gross structural estimates whether the protein is “mostly ordered” or “mostly disordered”, without residue-level resolution of disorder that would allow real functional inferences. Furthermore, these previous studies primarily focused on secreted calcium-binding phosphoproteins (SCPPs), i.e. vertebrate proteins involved in bone and teeth formation, which descended from the single ancestral gene encoding for osteonectin (SPARC) [19]. Because of the evolutionary relatedness of all these proteins, it cannot be ascertained if structural disorder is their inherited evolutionary trait or it is a structural feature mandatory for this type of function. This uncertainty in part results from invertebrate genomes being much less annotated than vertebrate (mammalian) ones, thus evolutionarily unrelated proteins of related function are neglected in these analyses.
To address structural disorder of proteins in biomineralization in general, we have applied bioinformatic predictions that provide residue level assessment, and found that almost all proteins which are closely related to HAP formation, regulation and orientation during growth are mostly or fully disordered. It is also shown that biomineralization and structural disorder of proteins not only correlate with the formation of vertebrate HAP but also the deposition of avian [44] and invertebrate calcium carbonate and diatom silica cell wall [45] structures. Due to the lack of evolutionary relatedness between vertebrate and non-vertebrate proteins, it can now be concluded that their extended structural disorder and special amino acid composition are inseparable from the emergence of ordered biomineral phase in a wide range of biological species. By applying the ensuing criteria of amino acid composition, we have also identified several novel proteins not yet implicated in biomineralization. By discussing these and all the other examples, we suggest that a novel type of functionality is apparent in this functional class of IDPs.
Section snippets
Methods
UniProtKB/Swiss-Prot database was filtered to match annotated keywords “biomineralization” and “secreted” and GO annotations: “ossification” (GO:0001503) and “biomineral tissue development” (GO:0031214). While the initial extraction was made without species exclusion, further studies focused on human proteins both to avoid redundancy and because these are better annotated. A few representative proteins from other species were also selected in the context of functional and evolutionary
Results
We have collected all proteins involved in biomineralization from humans and other mammals and also representative proteins involved in related processes, such as the formation of eggshell, mollusk shell and diatom cell wall (Supplementary Table S1). Key proteins, with their predicted disorder content and brief functional description, are summarized in Table 1. We found that human proteins linked to tooth and bone formation and the representative proteins from chicken, marine diatom and oyster
Discussion
In biomineralization, the stochastic chemical precipitation of minerals is turned into the orderly deposition of a crystal lattice in a biological context [23]. Together with cellular control, the process is regulated by a variety of hormones, transcription factors, enzymes and regulatory proteins, many of which have resisted structural characterization thus far. In fact, partial or full structural disorder of some proteins involved in biomineralization has been observed by NMR, CD and IR [18],
Acknowledgments
This research was supported by grants NK71582, CK80928 and PD83613 from the Hungarian Scientific Research Fund (OTKA), János Bolyai Research Scholarship of the Hungarian Academy of Sciences, a Korean-Hungarian Joint Laboratory grant from Korea Research Council of Fundamental Science and Technology (KRCF), TÁMOP-4.2.1/B-09/1/KMR-2010-0001 of the Hungarian National Development Agency, and an FP7 Marie Curie Initial Training Network grant (no. 264257, IDPbyNMR) and FP7 Infrastructures grant (no.
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