Use of complementary cation and anion heavy-atom salt derivatives to solve the structure of cytochrome P450 46A1

Abstract

Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the brain. The crystallization and heavy-atom structure solution of an active truncated CYP46A1 in complex with the high-affinity substrate analogue cholesterol-3-sulfate (CH-3S) is reported. The 2.6 angstroms structure of CYP46A1-CH-3S was solved using both anion and cation heavy-atom salts. In addition to the native anomalous signal from the haem iron, an NaI anion halide salt derivative and a complementary CsCl alkali-metal cation salt derivative were used. The general implications of the use of halide and alkali-metal quick soaks are discussed. The importance of using isoionic strength buffers, the titration of heavy-atom salts into different ionic species and the role of concentration are considered. It was observed that cation/anion-binding sites will occasionally overlap, which could negatively impact upon mixed RbBr soaks used for multiple anomalous scatterer MAD (MMAD). The use of complementary cation and anion heavy-atom salt derivatives is a convenient and powerful tool for MIR(AS) structure solution.

Figures

Figure 1

Typical tetragonal crystals of CYP46A1. The long tetragonal rods are approximately 40 µm in width and 300 µm in length. The red color is typical of the cytochrome P450s and is the consequence of optical absorbance by the haem prosthetic group. The cytochrome P450 46A1 packs in space group I4122 with the c axis parallel to the long axis of the rods.

Figure 2

The isomorphous and anomalous differences {χ2 ≃ [ΔI/σ(I)]2} from the three derivatives. The NaI (purple) and CsCl (green) derivatives both have a large solvent contribution at very low resolution. At the Cu Kα wavelength the anomalous Fe signal (red) is smaller than either of the isomorphous differences. The NaI data were limited to 2.8 Å resolution by the detector geometry. The χ2 values were calculated in SCALEPACK using 30 bins from 30 to 2.6 Å.

Figure 3

Patterson maps. These are xy Harker sections showing the asymmetric unit. Bijvoet difference Patterson maps at (a) z = 0.5 and (b) z = 0.25 are shown for the native data. Isomorphous difference Patterson maps at z = 0.5 are shown for (c) the NaI and (d) the CsCl quick-soak derivatives, respectively. Maps are contoured starting at 3σ in 0.5σ steps. Predicted Patterson peaks are labelled with a cross; observed peaks are labelled with the atom name. The iron anomalous and caesium isomorphous difference maps are at 2.6 Å resolution, while the iodide isomorphous derivative map is limited to 2.8 Å resolution. The origin peak at (1/2, 1/2, 1/2) has been removed.

Figure 4

Complementary cation and anion heavy-atom salt-derivative sites. Cross-eyed stereoview of the incomplete N-to-C-termini rainbow-colored CYP46A1 protein backbone and heavy-atom sites (colored spheres: I, purple; Cs, green) showing the nearby interacting residues as sticks. This figure was created using PyMOL (DeLano, 2003 ▶).

Figure 5

(a) Superposition of the 2.6 and 2.8 Å isomorphous difference cross-Fourier maps contoured at 3σ showing the interaction of caesium (green) or iodide (purple) ions with the corresponding CYP46A1 model (yellow or brown, respectively). Interactions are displayed as green dashes. Close caesium and iodide sites are observed near Arg76, which has moved in the NaI model (brown) and makes a contribution to the isomorphous difference signal (magenta mesh). The third interaction of Cs4, with the carbonyl of Asn388, is obscured in this view. (b) View of a twofold crystallographic symmetry-generated protein interface. The iodide (I8) sits on the twofold axis, flanked by four symmetry-related lysine side chains and two symmetry-related glycerol (GOL) molecules; the symmetry-generated molecules are colored lavender. This is one of four iodide sites that lacks hydrogen bonds; the lysine Nζ–I distance is 6 Å. This figure was drawn using XFIT/RASTER3D (Merritt & Bacon, 1997 ▶).