A solution NMR investigation into the murine amelogenin splice-variant LRAP (Leucine-Rich Amelogenin Protein)

Abstract

Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the (1)H-(15)N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the (1)H-(15)N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12 and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of (1)H-(15)N HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of (1)H-(15)N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full-length protein.

Fig. 1

Amino acid sequence of full-length amelogenin rp(H)M180 and the natural splice variant rp(H)LRAP. Underneath the sequence for rp(H)LRAP is a schematic illustration of identified regions in the proteins [19]. The N-terminal may be divided into three components: magenta = protein-protein interaction region, orange = linker region, and green = lectin-like binding tri-tyrosine domain. The C-terminal contains two regions: cyan = hydrophobic segment cleaved by enamelysin, and blue = C-terminal mineral-binding domain. The central majority of protein is enriched in the amino acids P, L, H, and Q and called the HQP-rich region. The two proteins are identical except for the absence of the lectin-like binding tri-tyrosine domain (green) and HQP-rich region in rp(H)LRAP.

Fig. 2

A) Overlay of the ¹H-¹⁵N HSQC spectrum of rp(H)M180 (red) and rp(H)LRAP (blue) under similar conditions (2% acetic acid, pH 3.0, 20°C) recorded at a ¹H resonance frequency of 750 (rp(H)LRAP) and 900 (rp(H)M180) MHz. B) Assignment of the amide resonance in the ¹H-¹⁵N HSQC spectra of rp(H) LRAP. In both figures the solid lines identify the two sets of cross peaks observed for the amide resonances between S12* and Y12.

Fig. 3

Overlay of the ¹H-¹⁵N HSQC spectrum of rp(H)LRAP in the absence of salt (cyan) and in the presence of a 540:1 molar ratio of NaCl:rp(H)LRAP. The tentative assignments of the amide resonances still remaining at the high salt concentration are shown.

Fig. 4

Summary of the results of the titration of NaCl into a solution of rp(H)LRAP in 2% acetic acid. The amide resonances that start to disappear or completely disappear in the ¹H-¹⁵N HSQC spectrum of ¹⁵N-labelled rp(H)LRAP are indicated by open and red circles, respectively. The NaCl:rp(H)LRAP molar ratios are indicated on the left. The top row (M2330) is the previously published results for rp(H)M180 at a NaCl:rp(H)M180 molar ratio of 2330:1 with the amide resonances that have begun to disappear or completely disappear in the ¹H-¹⁵N HSQC indicated by open and blue circles, respectively [17]. Underneath is a schematic illustration of the various regions of LRAP [19] (magenta = protein-protein interaction region, orange = linker, cyan = hydrophobic segment cleaved by enamelysin, blue = hydrophilic C-terminal mineral-binding domain).