Combined use of mass spectrometry and heterologous expression for identification of membrane-interacting peptides in cytochrome P450 46A1 and NADPH-cytochrome P450 oxidoreductase

Abstract

Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solution-exposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.

Figures

Fig. 1

The HPLC product profiles of the extracts from the enzyme assays carried out with (a) CYP46A1-containing E. coli membranes; (b) boiled CYP46A1-containing E. coli membranes; and (c) purified recombinant CYP46A1. Assay conditions were as described in Materials and Methods.

Fig. 2

Enzymatic reduction of purified recombinant CYP46A1 with NADPH and CPR-containing E. coli membranes (solid line) and with sodium dithionite (dashed line). Assay conditions were as described in Materials and Methods.

Fig. 3

MS spectra of the extracts from the trypsin- (a) and chymotrypsin-treated (c) CYP46A1-containing E. coli membrane. Peptides that correspond to CYP46A1 are labeled and those from E. coli are not. Enlarged MS spectrum of the tryptic peptide ion with m/z [M+H]+ value 2716.41 is shown in panel (b). The MS/MS spectrum of the chymotryptic peptide ion with m/z [M+H]+ value 972.56 is shown in panel (d) and has b1 (R), b3 (RLV), b7 (RLVPGQR), y2 (RF), y5 (PGQRF), y5-NH3 (loss of ammonia from y5), and internal fragment (PGQ) ions that match the RLVPGQRF sequence.

Fig. 4

MS spectra of the extracts from the trypsin- (a) and chymotrypsin-treated (c) CPR-containing E. coli membrane. Peptides that correspond to CPR are labeled and those from E. coli are not. The MS/MS spectrum of the tryptic peptide ion with m/z [M+H]+ value 1637.85 is shown in panel (b) and has b2 (TA), b3 (TAL), b8 (TALTYYLD), y1 (R), y3 (PPR), y4 (NPPR), y5 (TNPPR), y6 (ITNPPR), y7 (DITNPPR), y8 (LDITNPPR), y9 (YLDITNPPR), and y10 (YYLDITNPPR) fragment ions that match the TALTYYLDITNPPR sequence. The MS/MS spectrum of the chymotryptic peptide ion with m/z [M+H]+ value 1137.55 is shown in panel (d) and has b2 (MG), b3 (MGF), b4 (MGFI), b8 (MGFIQERA) y1 (W), y2 (AW), y3 (RAW), y4 (ERAW), y5 (QERAW), and y6 (IQERAW) fragment ions that match the MGFIQERAW sequence.

Fig. 5

The proposed membrane orientation of CYP46A1. Black horizontal line separates the cytosol (above) and the lipid bilayer (below). The CYP46A1 molecule (PDB ID 2Q9F) is shown in four different views rotated counterclockwise by 90°. (a) Spatial position of the heme (shown as sticks and balls in red), substrate (cholesterol sulfate, shown as sticks and balls in lime), the missed cleavage sites (shown as sticks in magenta), and amino acid residues flanking the putative membrane-interacting peptides (shown as sticks in blue). The N-terminus, which connects to the transmembrane anchor, is shown as orange ball. (b) The surface representation of CYP46A1. The views and coloring are the same as in (a); the putative membrane-interacting peptides are in green.

Fig. 6

The proposed membrane orientation of CPR. Black horizontal line separates the cytosol (above) and the lipid bilayer (below). The CPR molecule (PDB ID 1AMO) is shown in four different views rotated counterclockwise by 90°. (a) Spatial position of NADP(H) (shown as sticks and balls in green), FAD (shown as sticks and balls in yellow), FMN (shown as sticks and balls in red), the missed cleavage sites (shown as sticks in magenta), and amino acid residues flanking the putative membrane-interacting peptides (shown as sticks in blue). The N-terminus, which connects to the transmembrane segment, is shown as orange ball. (b) The surface representation of CPR. The views and coloring are the same as in (a); the putative membrane-interacting peptides are in green. (c) The surface representation of CPR showing the domain structure; the FAD- and NADPH-binding domains are in aquamarine, the connecting domain is in yellow, and the FMN-binding domain is in violet. Q150 and R313 (Q153 and R316 in human CPR) whose mutations result in disordered steroidogenesis are in black.

Fig. 7

The HPLC product profiles of the extracts from the enzyme assays carried out with (a) purified CYP46A1 and cholesterol-enriched E. coli membranes; (b) control incubation with boiled CYP46A1; and (c) purified recombinant CYP46A1 and cholesterol. Assay conditions were as described in Materials and Methods.