Molecular Characterization of the 30-AA N-Terminal Mineral Interaction Domain of the Biomineralization Protein AP7

Abstract

The AP7 protein is one of several mollusk shell proteins which are responsible for aragonite polymorph formation and stabilization within the nacre layer of the Pacific red abalone, H. rufescens. Previously, we demonstrated that the 30-AA N-terminal domain of AP7, denoted as AP7-1, exists as an unfolded sequence and possesses the capability of inhibiting calcium carbonate crystal growth in vitro via growth step frustration or interruption. However, very little is known with regard to the interactive capabilities of this sequence with Ca(II) and with calcium carbonates. Using multidisciplinary techniques, we determine that the AP7-1 polypeptide interacts with Ca(II) ions at the -DD- sequence clusters, yet retains its unfolded, conformationally labile structure in the presence of Ca(II) ions. Further, NMR experiments reveal that the extended structured sequence blocks, -GNGM-, -SVRTQG-, and -ISYL, exhibit motional, chemical exchange, and/or backbone geometry perturbations in response to Ca(II) interactions with AP7-1. Solid-state NMR magic angle spinning studies verify that during the course of in vitro calcium carbonate crystal growth, AP7-1 becomes bound to calcite fragments and cannot be entirely displaced from the mineral fragments using competitive Ca(II) washing. Finally, using a scrambled sequence version of the AP7-1 polypeptide, we observe that sequence scrambling does not adversely affect the crystal growth inhibitory activity of AP7-1, suggesting that the amino acid composition of AP7-1 may be more critical to growth step inhibition than the linear ordering of amino acids.

Intrinsically Disordered Proteins in Biomineralization

“…Mammals DMP1 4.0 CD, DLS, FTIR, SAXS (Gericke et al, 2010;He et al, 2003aHe et al, , 2003b DPP 2.8 CD, NMR, SAXS (Cross et al, 2005;Evans et al, 1994;Fujisawa & Kuboki, 1998;George & Hao, 2005;He et al, 2005b;Lee et al, 1977) BSP 4.1 CD, NMR, SAXS (Fisher et al, 2001;Tye et al, 2003Tye et al, , 2005Wuttke et al, 2001) OPN 4.4 CD, NMR, FTIR (Fisher et al, 2001;Gorski et al, 1995) amelogenin 6.6 CD, NMR (Buchko et al, 2010;Delak et al, 2009b;Ndao et al, 2011;Shaw et al, 2008) statherin 8.0 CD, NMR (Long et al, 2001;Naganagowda et al, 1998;Raj et al, 1992) lithostathine 5.7 CD (Gerbaud et al, 2000) Haliotis rufescens AP7 5.2 CD, NMR (Kim et al, 2004(Kim et al, , 2006aMichenfelder et al, 2003;Wustman et al, 2004) Atrina rigita Asprich 2.7-3.5 CD, NMR (Collino et al, 2006;Delak et al, 2009aDelak et al, , 2008Kim et al, 2008;Ndao et al, 2010) Procambrus clarkii CAP-1 3.9 CD (Inoue et al, 2007) Strongylocentrotus purpuratus SM50 10.8 CD, NMR (Xu & Evans, 1999;Zhang et al, 2000) PM27 8.1 CD, NMR Danio rerio Stm 4.1 CD, gel filtration (Kaplon et al, 2008(Kaplon et al, , 2009 Table 1. IDPs involved in biomineralization of calcium carbonate and phosphate for which a disordered structure has been confirmed...…”

“…(Michenfelder et al, 2003;Wustman et al, 2004), while the C-terminus is -helical (Kim et al, 2006a). Interestingly, AP7's C-terminus end did not influence crystal growth (Kim et al, 2006a), while AP7's N-terminus was responsible for interactions with calcium ions and led to an inhibition of calcite growth in vitro (Kim et al, 2004;Michenfelder et al, 2003). It should be noted that full-length AP7 protein had the highest effect on calcite crystal growth (Kim et al, 2006a).…”

“…This observation suggests that the flexibility and plasticity of IDPs might be crucial factors which inhibit crystal growth, although the well-defined, ordered secondary structure may also be necessary. It should be noted that high concentrations of calcium ions led to the precipitation of AP7's N-terminus, which may indicate calcium-dependent oligomerization (Kim et al, 2004). Finally, full-length AP7 undergoes a conformational change to an -helical structure as a function of TFE concentration (Amos & Evans, 2009).…”

“…The analysis of n16's primary sequence revealed the requisite amino acid residues associated with a calcium carbonate modification domain within the C-and N-terminae of the mature protein (n16-C and n16-N, respectively) (Kim et al, 2004). Both sequences modify the morphology of calcium carbonate crystals and induce calcite growth in vitro (Kim et al, 2004).…”

Probing the Organic-Mineral Interface at the Molecular Level in Model Biominerals

“…This approach has been used to examine the participation of a number of polypeptides within biomineral nucleation and crystal growth assay systems. [9][10][11][12][13][14][15][16][17] Although limited in their scope, such in vitro systems provide much needed information on the role of proteins within mineralizing systems, and can serve as a stepping stone towards more complex model systems that possess larger numbers of molecular participants.…”