Rachel E. Helbling, Walter Aeschimann, Fabio Simona, Achim Stocker, Michele Cascella

We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of [Formula: see text]-tocopherol transfer protein ([Formula: see text]-TTP) towards [Formula: see text]-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for [Formula: see text]-tocopherol to [Formula: see text]-TTP 8.26[Formula: see text]2.13 kcal mol[Formula: see text] lower than that of [Formula: see text]-tocopherol. Our calculations show that [Formula: see text]-tocopherol binds to [Formula: see text]-TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of [Formula: see text]-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to [Formula: see text]-tocopherol, with striking structural similarities to the wild-type-[Formula: see text]-tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of [Formula: see text]-TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover, the identification of a [Formula: see text]-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for [Formula: see text]-tocopherol selection in omnivorous animals.