Ligand‐induced conformational changes in cytochrome P450 3A4 detected by luminescence resonance energy transfer (LRET)

Abstract

Recent advances in solving crystal structures of cytochromes P450 have revealed a remarkable plasticity that allows the enzymes to accommodate substrates of different shapes and sizes. However, the data on conformational mobility of cytochromes P450 in solution are limited, and the role of conformational plasticity in their allosteric behavior remains obscure. The major human drug‐metabolizing cytochrome P450 3A4 (CYP3A4), exhibits both homo‐ and heterotropic cooperativity. We probed ligand‐induced conformational changes in the enzyme with LRET. For this purpose we produced a number of mutants containing only two cysteine residues accessible to modification. Unchanged ligand binding properties of the Cys64/Cys468 mutant prompted its use in the further studies. Successive labeling of this mutant with the phosphorescent dye erythrosine and a far‐red fluorophore DY‐731 allowed us to create an intramolecular pair of LRET donor and acceptor. This double‐labeled protein was used to probe the conformational response of the enzyme upon interactions with a series of ligands. The changes in the efficiency of LRET induced by the substrates 1‐pyrenebutanol and bromocriptine were negligible. In contrast, a pronounced effect of such ligands as α‐naphtophlavone or quinidine reveals a conformational transition induced by these heterotropic activators. (Supported by GM054995).