Abstract
To accomplish key physiological processes ranging from drug metabolism to steroidogenesis, human microsomal cytochrome P450 enzymes require the sequential input of two electrons delivered by the FMN domain of NADPH-cytochrome P450 reductase. Although some human microsomal P450 enzymes can instead accept the second electron from cytochrome b5, for human steroidogenic CYP17A1, the cytochrome P450 reductase FMN domain delivers both electrons, and b5 is an allosteric modulator. The structural basis of these key but poorly understood protein interactions was probed by solution NMR using the catalytically competent soluble domains of each protein. Formation of the CYP17A1·FMN domain complex induced differential line broadening of the NMR signal for each protein. Alterations in the exchange dynamics generally occurred for residues near the surface of the flavin mononucleotide, including 87-90 (loop 1), and for key CYP17A1 active site residues. These interactions were modulated by the identity of the substrate in the buried CYP17A1 active site and by b5. The FMN domain outcompetes b5 for binding to CYP17A1 in the three-component system. These results and comparison with previous NMR studies of the CYP17A1·b5 complex suggest a model of CYP17A1 enzyme regulation.
Highlights
Because the different labeled proteins have different intrinsic dynamics, the temperatures at which different experiments were undertaken were empirically determined as those that best revealed changes upon titration
Summary of P450/Ligand/CPR/b5 System under Study—In humans, microsomal NADPH-cytochrome P450 reductase and cytochrome b5 proteins interact with cytochrome P450 enzymes, all three integral membrane proteins are anchored on the surface of the endoplasmic reticulum
This approach is supported by NMR and x-ray experiments previously demonstrating that the structure of the isolated FMN domain is very similar to its structure when part of the larger multidomain CPR and regardless of which conformational state the CPR adopts [3,4,5, 26]
Summary
Protein Expression and Purification—DNA encoding the human CPR FMN binding domain (residues 62–241) fused to a C-terminal His tag was synthesized and inserted into the pET15b expression vector (GenScript, Piscataway, NJ). Eluted protein was pooled, concentrated to 1 ml using an Amicon centrifugal filter unit (EMD Millipore, Darmstadt, Germany), and loaded on a Superdex 200 resin (GE Healthcare) This column had been pre-equilibrated with 20 mM Tris-HCl, 0.3 M NaCl, 5 M FMN, 0.2 mM dithiothreitol, 0.02 mM EDTA, pH 7.9, and subsequently run with the same buffer for 1.5 column volumes at 1 ml/min. Partial assignment of the CYP17A1 backbone was carried out using a combination of 1H-15N TROSY HSQC [36] spectra with selective labeling of particular residues (Leu, Val, Ile, Ala, and Phe) and data from the three-dimensional experiments TRHNCA [37, 38], TRHNCACB [38], TRHNCACO [38], and N-NOESY of CYP17A1 in complex with abiraterone
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