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1. The flexible structure of the K24S28 region of Leucine-Rich Amelogenin Protein (LRAP) bound to apatites as a function of surface type, calcium, mutation, and ionic strength

Author

Junxia eLu, Sarah D Burton, Yimin Sharon Xu, Garry W. Buchko, and Wendy J Shaw

Subjects
Amelogenin, secondary structure, Solid state NMR, LRAP, biomineralization protein, Physiology, QP1-981
Abstract

Leucine-Rich Amelogenin Protein (LRAP) is a member of the amelogenin family of biomineralization proteins, proteins which play a critical role in enamel formation. Recent studies have revealed the structure and orientation of the N- and C-terminus of LRAP bound to hydroxyapatite (HAP), a surface used as an analog of enamel. The structure of one region, K24 to S28, was found to be sensitive to phosphorylation of S16, the only naturally observed site of serine phosphorylation in LRAP, suggesting that K24S28 may sit at a key region of structural flexibility and play a role in the protein’s function. In this work, we investigated the sensitivity of the structure and orientation of this region when bound to HAP as a function of several factors which may vary during enamel formation to influence structure: the ionic strength (0.05 M, 0.15 M, 0.2 M), the calcium concentration (0.07 mM and 0.4 mM), and the surface to which it is binding (HAP and carbonated apatite (CAP), a more direct mimic of enamel). A naturally occurring mutation found in amelogenin (T21I) was also investigated. The structure in the K24S28 region of the protein was found to be sensitive to these conditions, with the CAP surface and excess Ca2+ (8:1 [Ca2+]:[LRAP-K24S28(+P)]) resulting in a tighter helix, while low ionic strength relaxed the helical structure. Higher ionic strength and the point mutation did not result in any structural change in this region. The distance of the backbone of K24 from the surface was most sensitive to excess Ca2+ and in the T21I-mutation. Collectively, these data suggest that phosphorylated LRAP is able to accommodate structural changes while maintaining its interaction with the surface, and provides further evidence of the structural sensitivity of the K24S28 region, a sensitivity that may contribute to function in biomineralization.

Published
2014
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2. Phosphorylation and Ionic Strength Alter the LRAP–HAP Interface in the N-Terminus

Author

Jun-xia Lu, Wendy J. Shaw, and Yimin Sharon Xu

Subjects
Models, Molecular, Protein Conformation, Molecular Sequence Data, Plasma protein binding, Biochemistry, Article, Mice, Protein structure, Dental Enamel Proteins, stomatognathic system, Dynamic light scattering, Animals, Amino Acid Sequence, Phosphorylation, skin and connective tissue diseases, Nuclear Magnetic Resonance, Biomolecular, Amelogenin, Enamel paint, Chemistry, Osmolar Concentration, stomatognathic diseases, Crystallography, Durapatite, Ionic strength, visual_art, visual_art.visual_art_medium, sense organs, Crystallite, Protein Binding
Abstract

The conditions present during enamel crystallite development change dramatically as a function of time, including the pH, protein concentration, surface type, and ionic strength. In this work, we investigate the role that two of these changing conditions, pH and ionic strength, have in modulating the interaction of the amelogenin, LRAP, with hydroxyapatite (HAP). Using solid-state NMR dipolar recoupling and chemical shift data, we investigate the structure, orientation, and dynamics of three regions in the N-terminus of the protein: L(15) to V(19), V(19) to L(23), and K(24) to S(28). These regions are also near the only phosphorylated residue in the protein pS(16); therefore, changes in the LRAP-HAP interaction as a function of phosphorylation (LRAP(-P) vs LRAP(+P)) were also investigated. All of the regions and conditions studied for the surface immobilized proteins showed restricted motion, with indications of slightly more mobility under all conditions for L(15)(+P) and K(24)(-P). The structure and orientation of the LRAP-HAP interaction in the N-terminus of the phosphorylated protein is very stable to changing solution conditions. From REDOR dipolar recoupling data, the structure and orientation in the region L(15)V(19)(+P) did not change significantly as a function of pH or ionic strength. The structure and orientation of the region V(19)L(23)(+P) were also stable to changes in pH, with the only significant change observed at high ionic strength, where the region becomes extended, suggesting this may be an important region in regulating mineral development. Chemical shift studies also suggest minimal changes in all three regions studied for both LRAP(-P) and LRAP(+P) as a function of pH or ionic strength, and also reveal that K(24) has multiple resolvable resonances, suggestive of two coexisting structures. Phosphorylation also alters the LRAP-HAP interface. All of the three residues investigated (L(15), V(19), and K(24)) are closer to the surface in LRAP(+P), but only K(24)S(28) changes structure as a result of phosphorylation, from a random coil to a largely helical structure, and V(19)L(23) becomes more extended at high ionic strength when phosphorylated. These observations suggest that ionic strength and dephosphorylation may provide switching mechanisms to trigger a change in the function of the N-terminus during enamel development.

Published
2013

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