Structure, Orientation, and Dynamics of the C-Terminal Hexapeptide of LRAP Determined Using Solid-State NMR

Abstract

Amelogenin is the predominant protein found during enamel development and has been shown to be essential to proper enamel formation. Leucine-rich amelogenin peptide (LRAP) is a naturally occurring splice variant that preserves the charged N-and C-termini of full length amelogenin, regions thought to be crucial in interacting with hydroxaypatite. Particularly, the highly charged Cterminal hexapeptide (KREEVD) is thought to be the region most intimately interacting with hydroxyapatite (HAP). The structure of this charged region was investigated, along with the proximity to the surface and the mobility of two of the residues. The structure was found to be consistent with a random coil or more extended structure, as has been seen for more internalized residues in the C-terminus. The backbone K 54 ( 13 C′), V 58 ( 13 C′), and V 58 ( 15 N) were all found to be close to the surface of HAP, ∼ 6.0 Å from the nearest 31 P atom, suggesting a strong interaction and emphasizing the importance of these residues in interacting with HAP. However, both ends of the hexapeptide at residues K 54 and V 58 experience significant mobility under hydrated conditions, implying that another portion of the protein helps to stabilize the strong LRAP-HAP interaction. Interestingly, the backbone of the C-terminal third of the protein is consistently 6.0 Å from the HAP surface, providing a model in this region of the protein lying flat on the surface with no threedimensional folding. The combination of these features, that is, a random coil structure, a significant mobility, and a lack of three-dimensional folding in this region of the protein, may have an important functional role, possibly allowing maximum crystal inhibition at low protein concentrations.

Monomeric Amelogenin’s C-Terminus Modulates Biomineralization Dynamics of Calcium Phosphate

“…39,40 These clusters co-assemble as chains of nanoparticles that evolve into long co-aligned crystals resembling those found in biological enamel. 41 NMR spectroscopy studies 34,42 and others 43,44 have provided direct evidence that the Amel C-terminus is close enough to the HAP surface to direct growth. Our previous studies 40,41 have concluded that hydrophilic C-termini toward the exterior, away from the primarily hydrophobic cores of a full-length Amel protein, 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 …”

“…33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 14 have an important functional role, possibly allowing maximum crystal inhibition at low protein concentrations. 34 Tarasevich et al demonstrated that LRAP is primarily a monomer in saturated calcium phosphate solutions. 35 Moreover, the LRAP protein is not completely extended on the surface, having some degree of structure away from the surface by neutron reflectivity.…”

Mineral Association Changes the Secondary Structure and Dynamics of Murine Amelogenin

“…Other evidence of motion is described below. The observation of some protein mobility suggests that the interface between amelogenin and HAP in the lysine regions allows for flexibility, an observation made for regions of both LRAP and statherin bound to HAP (Shaw et al, 2000;Shaw and Ferris, 2008) that may be related to function, such as allowing a single protein to cover more growth sites or serving a second function, such as protein binding.…”

“…This would result if part of the amelogenin molecule interacts strongly with the HAP surface (for instance, the C-terminus), while the other part (for instance, the N-terminus) does not. Analysis of previous SSNMR data with LRAP showed that both the N-and C-termini interacted tightly with HAP surfaces, whether phosphorylated at S16 or not, although the N-terminal K24 residue was slightly farther from the surface (7.5 Å to 9.0 Å) and became less structured for the non-phosphorylated sample (Shaw et al, 2004;Shaw and Ferris, 2008;Masica et al, 2011). The recombinant sample reported here is also non-phosphorylated, and it is possible that a looser interaction of the N-terminus could result in a similar increase in mobility and loss of structure.…”

“…Solid-state NMR (SSNMR) spectroscopy is uniquely suited to the study of the structure of immobilized proteins, as demonstrated by dipolar recoupling studies that determined the structure and orientation of the amelogenin splicevariant LRAP bound to HAP (Shaw and Ferris, 2008;Masica et al, 2011;Lu et al, 2013). To date, studies of biomineralization proteins have been limited to small proteins (< ~60 residues).…”