3-DIMENSIONAL MODELING OF HUMAN CYTOCHROME-P450 1A2 AND ITS INTERACTION WITH CAFFEINE AND MEIQ

The three-dimensional modelling of proteins is a useful tool to fill t he gap between the number of sequenced proteins and the number of expe rimentally known 3D structures. However, when the degree of homology b etween the protein and the available 3D templates is low, model buildi ng becomes a difficult task and the reliability of the results depends critically on the correctness of the sequence alignment. For this rea son, we have undertaken the modelling of human cytochrome P450 1A2 sta rting by a careful analysis of several sequence alignment strategies ( multiple sequence alignments and the TOPITS threading technique). The best results were obtained using TOPITS followed by a manual refinemen t to avoid unlikely gaps. Because TOPITS uses secondary structure pred ictions, several methods that are available for this purpose (Levin, G ibrat, DPM, NnPredict, PHD, SOPM and NNSP) have also been evaluated on cytochromes P450 with known 3D structures. More reliable predictions on alpha-helices have been obtained with PHD, which is the method impl emented in TOPITS. Thus, a 3D model for human cytochrome P450 1A2 has been built using the known crystal coordinates of P450 BM3 as the temp late. The model was refined using molecular mechanics computations. Th e model obtained shows a consistent location of the substrate recognit ion segments previously postulated for the CYP2 family members. The in teraction of caffeine and a carcinogenic aromatic amine (MeIQ), which are characteristic P450 1A2 substrates, has been investigated. The sub strates were solvated taking into account their molecular electrostati c potential distributions. The docking of the solvated substrates in t he active site of the model was explored with the AUTODOCK programme, followed by molecular mechanics optimisation of the most interesting c omplexes. Stable complexes were obtained that could explain the oxidat ion of the considered substrates by cytochrome P450 1A2 and could offe r an insight into the role played by water molecules.