Structural Analysis of Breast-Milk αS1-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation

Breast-milk alpha(S1)-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated alpha(S1)-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of alpha(S1)-casein indicated high alpha-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the alpha-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the alpha-helical content was significantly reduced, similar to what it was after incubation at 80 degrees C. This conformation showed no in vitro induction of IL-8 secretion via TLR4. A synthetic peptide corresponding to V-77-E-92 of alpha(S1)-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that alpha(S1)-casein appears in two distinct conformations, an alpha-helical TLR4-agonistic and a less alpha-helical TLR4 non-agonistic conformation induced by phosphorylation. This is to indicate that the immunomodulatory role of alpha(S1)-casein, as described before, could be regulated by conformational changes induced by phosphorylation.