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

Abstract

We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of α-tocopherol transfer protein (α-TTP) towards α-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for α-tocopherol to α-TTP 8.26±2.13 kcal mol-1 lower than that of γ-tocopherol. Our calculations show that γ-tocopherol binds to α-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 α-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to γ-tocopherol, with striking structural similarities to the wild-type-α-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 α-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 γ-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for α-tocopherol selection in omnivorous animals. © 2012 Helbling et al.