Engineering Tocopherol Selectivity in α-TTP: A Combined In Vitro/In Silico Study

We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of alpha-tocopherol transfer protein (alpha-TTP) towards alpha-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for alpha-tocopherol to alpha-TTP 8.26 +/- 2.13 kcal mol(-1) lower than that of gamma-tocopherol. Our calculations show that gamma-tocopherol binds to alpha-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 alpha-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to gamma-tocopherol, with striking structural similarities to the wild-type-alpha-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 alpha-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 gamma-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for alpha-tocopherol selection in omnivorous animals.