Delineation of Conformational Preferences in Human Salivary Statherin by ¹H, ³¹P NMR and CD Studies: Sequential Assignment and Structure-Function Correlations

Abstract

Membrane—induced solution structure of human salivary statherin, a 43 amino acid residue acidic phosphoprotein, has been investigated by two—dimensional proton nuclear magnetic resonance (2D ¹H NMR) spectroscopy. NMR assignments and structural analysis of this phosphoprotein was accomplished by analyzing the pattern of sequential and medium range NOEs, αCH chemical shift perturbations and deuterium exchange measurements of the amide proton resonances. The NMR data revealed three distinct structural motifs in the molecule: (1) an α-helical structure at the N-terminal domain comprising Asp¹-Tyr¹⁶, (2) a polyproline type II (PPII) conformation predominantly occurring at the middle proline-rich domain spanning Gly¹⁹-Gln³⁵, and (3) a 3₁₀−helical structure at the C-terminal Pro³⁶-Phe⁴³ sequence. Presence of a few weak d_αN(i,i+2) suggests that N-terminus also possesses minor population of 3₁₀−helical conformation. Of the three secondary structural elements, helical structure formed by the N-terminal residues, Asp¹-Ile¹¹ appears to be more rigid as observed by the relatively very slow exchange of amide hydrogens of Glu⁵-Ile¹¹. ³¹P NMR experiments clearly indicated that N-terminal domain of statherin exists mainly in disordered state in water whereas, upon addition of structure stabilizing co-solvent, 2,2,2-trifluo-rethanol (TFE), it showed a strong propensity for helical conformation. Calcium ion interaction studies suggested that the disordered N-terminal region encompassing the two vicinal phosphoserines is essential for the binding of calcium ions in vivo. Results from the circular dichroism (CD) experiments were found to be consistent with and complimentary to the NMR data and provided an evidence that non-aqueous environment such as TFE, could induce the protein to fold into helical conformation. The findings that the statherin possesses blended solvent sensitive secondary structural elements and the requirement of non-structured N-terminal region under aqueous environment in calcium ion interaction may be invaluable to understand various physiological functions of statherin in the oral fluid.