Molecular dynamics simulations of the holo and apo forms of retinol binding protein. Structural and dynamical changes induced by retinol removal.

The effects of removing retinol from the X-ray structure of holo-retinol binding protein are studied using the molecular dynamics technique. Structural and dynamical properties emerging from an 80 ps simulation of the apo form, for which no crystallographic structure is available, are compared with the results of a 70 ps trajectory of the holo-protein. Dynamical stationarity is attained after roughly 30 ps, and the resulting average structure is proposed as a reasonable model of the apo-protein. Conformational changes are observed for the loops at the beta-barrel entrance during the non-equilibrium part of the apo-trajectory. Tryptophan labelling experiments and retinoid reconstitution experiments point towards this part of the molecule as being involved in prealbumin binding. Structural changes in this region may therefore explain the differences in prealbumin affinity between the apo and holo forms. Furthermore, a change in the position of the alpha-helix, corresponding to a pivot around its C terminus, is observed for the apo-protein. The resulting conformation of the alpha-helix is found to be similar to that in apo-beta-lactoglobulin, which also can bind retinol and for which a crystal structure exists. The results from the holo simulation are compared to the crystallographic data and show good agreement. The dynamics of the secondary and tertiary structural elements are analysed and compared for the two forms. The beta-barrel is found to be extremely cooperative in its atomic motions in both simulations, and the top and bottom beta-sheets perform collective fluctuations with respect to each other in the low-frequency limit of the simulations. The dynamics of the alpha-helical region presents clear differences between the two forms; while the holo-protein has a well-defined spectrum for the longitudinal stretching mode, the apo form displays a fairly large bending of the alpha-helix at several points of the trajectory.