AP7 is an extracellular aragonite-associated protein of the nacre layer of the mollusk Haliotis rufescens and possesses a 36-amino acid C-terminal domain that exhibits sequence homology to the C subclass of the RING domain intracellular protein family. We report here novel findings which implicate AP7 as a member of the intrinsically disordered protein class (IDP) and reveal new aspects of AP7 mineralization activity. AP7 is partially disordered but can undergo additional folding in the presence of TFE. AP7 binds Zn(II) ions in a non-tetracoordinate complex but does not require Zn(II) either for folding or for in vitro function. In addition to limiting calcite crystal growth, AP7 is also observed to induce aggregate formation within in vitro mineralization assays, and these aggregates are either amorphous (type A) or crystalline (type B) in appearance. The type A aggregate displays an irregular morphology and round, dark, electron dense deposits that do not give rise to a diffraction pattern. In contrast, the type B aggregates possess either organized parallel crystal clusters or highly dense hexagonal clusters that are confirmed by electron diffraction to be aragonite. This stabilization of aragonite is remarkable in that it occurred in the presence of AP7 alone and did not require typical aragonite stabilization agents such as Mg(II), other nacre proteins, or an organized organic matrix. The ability of a partially disordered C-RING protein to perform inorganic phase stabilization represents a new twist on both the RING domain and IDP stories, and this process of aggregate formation may provide an important clue with regard to the protein-mediated nacre formation process.
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