Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET

Abstract

A cysteine was introduced into the FG-loop (P187C) of CYP51 from Mycobacterium tuberculosis (MT) for selective labeling with BODIPY and fluorescence energy transfer (FRET) analysis. Forster radius for the BODIPY-heme pair was calculated assuming that the distance between the heme and Cys187 in solution corresponds to that in the crystal structure of ligand free MTCYP51. Interaction of MTCYP51 with azole inhibitors ketoconazole and fluconazole or the substrate analog estriol did not influence the fluorescence, but titration with the substrate lanosterol quenched BODIPY emission, the effect being proportional to the portion of substrate-bound MTCYP51. The detected changes correspond to ~10 Å decrease in the calculated distance between BODIPY-Cys187 and the heme. The results confirm 1) functional importance of conformational motions in the MTCYP51 F/G segment and 2) applicability of FRET to monitor them in solution. KeywordsCytochrome P450; sterol 14α-demethylase; ligand binding; conformational changes; FRET The cytochrome P450 superfamily currently includes more than 6,300 enzymes (http://drnelson.utmem.edu/CytochromeP450.html) oxidizing a wide variety of xenobiotics (compounds from the environment) and endogenous substrates. Even at less than 16% amino acid identity, all P450s preserve a common overall structural fold (α-helical with orthogonal bundle architecture according to CATH classification [1]) and the set of secondary structural elements that allow the superfamily to metabolize a wide variety of diverse and unrelated structures. By now it has become quite generally accepted that this enormous catalytic versatility arises from the P450 conformational flexibility [2,3]. Ligandinduced conformational changes encompassing small to large movements are seen in the Xray structures of several bacterial and drug-metabolizing mammalian CYPs 1 (e.g. CYPs101 * Corresponding author: Michael R. Waterman, Tel.: 615-343-1373; Fax: 615-322-4349; E-mail: michael.waterman@vanderbilt.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. . On the other hand, site-directed mutagenesis of five amino acid residues in this region (L172, G175, P178, R194 and D195), which are highly conserved in the CYP51 family and exposed into the substrate binding cavity of MTCYP51 has shown that all of them are essential for MTCYP51 catalytic activity at the stage of the interaction with substrate. Moreover, mutation of A197 in the middle of the MTCYP51 G-helix to the helix-breaking glycine causes about one order of magnitude enzyme activation and sharp increase in the substrate bound (high-spin) po...

Binding of a physiological substrate causes large-scale conformational reorganization in cytochrome P450 51

“…The lack of conformational changes in response to inhibitor binding in the CYP51 crystal structures is in a good agreement with our previous FRET-based data (in the solution) that detected no conformational changes in the CYP51 from Mycobacterium tuberculosis upon binding of ketoconazole, fluconazole, or the substrate analog estriol (16). The same experiments, however, demonstrated up to 10 Å movement in the enzyme FG arm (FЉ helix) in response to the binding of the substrate lanosterol, thus strongly suggesting that large-scale conformational dynamics must still be involved in CYP51 catalysis, and the evidence for this is provided for the first time in this study.…”

Structural differences between soluble and membrane bound cytochrome P450s

“…This was proposed for many soluble cytochromes P450 based on available X-ray structures of CYP101 [16-17], CYP158A2 [47], CYP107L1 [44], CYP113 [45], and CYP51 [48]. However, in cytochromes P450 that are membrane bound this loop connecting F and G helices is extended and usually includes additional F’ and / or G’ helices, which interact with and are inserted into the lipid bilayer.…”

Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) from Aspergillus fumigatus and Molecular Basis for the Development of Antifungal Drugs

“…SRS1 represents the major CYP51 substrate binding area. It carries the family signature 1 (13), the conserved or phylumspecific residues that are essential for enzyme function (37,51,52). In the T. brucei CYP51 complex with the substrate analog MCP, this area of the protein (shown as a yellow ribbon in Fig.…”