Steroid and protein ligand binding to cytochrome P450 46A1 as assessed by hydrogen-deuterium exchange and mass spectrometry

Abstract

Cytochrome P450 46A1 (CYP46A1) is a key enzyme responsible for cholesterol elimination from the brain. This P450 can interact with different steroid substrates and protein redox partners. We utilized hydrogen-deuterium (H-D) exchange mass spectrometry for investigating CYP46A1-ligand interactions. First, we tested the applicability of the H-D exchange methodology and assessed the amide proton exchange in substrate-free and cholesterol-sulfate-bound P450. The results showed good correspondence to the available crystal structures and prompted investigation of the CYP46A1 interactions with the two steroid substrates cholesterol and 24S-hydroxycholesterol and the protein redox partner adrenodoxin (Adx). Compared to substrate-free P450, four peptides in cholesterol-bound CYP46A1 (65-80, 109-116, 151-164, and 351-361) and eight peptides in 24S-hydroxycholesterol-bound enzyme (50-64, 65-80, 109-116, 117-125, 129-143, 151-164, 260-270, and 364-373) showed altered deuterium incorporation. Most of these peptides constitute the enzyme active site, whereas the 351-361 peptide is from the region putatively interacting with the redox partner Adx. This also defines the proximal (presumably water) channel that opens in CYP46A1 upon substrate binding. Reciprocal studies of Adx binding to substrate-free and cholesterol-sulfate-bound CYP46A1 revealed changes in the deuteration of the Adx-binding site 144-150 and 351-361 peptides, active site 225-239 and 301-313 peptides, and in the 265-276 peptide, whose functional role is not yet known. The data obtained provide structural insights into how substrate and redox partner binding are coordinated and linked to the hydration of the enzyme active site.

Figures

Figure 1

Sequence coverage of human CYP46A1 by peptic digestion. The peptides considered suitable for obtaining H-D exchange data are indicated by arrows and cover 83% of the (Δ2–50) CYP46A1 sequence. The secondary structural elements are shown above the sequence. Helixes and sheets are shown in red and blue, respectively.

Figure 2

Peptides showing altered H-D exchange upon binding of cholesterol sulfate. The scale of the y axis shows the expected number of deuterated amide groups in each peptide. Adjustment for the back-exchange was not performed; therefore, the difference in deuterium incorporation between substrate-free (○) and cholesterol sulfate-bound (■) CYP46A1 is a relative value.

Figure 3

Regions in the CYP46A1 structure showing altered deuterium incorporation upon binding of cholesterol sulfate. Substrate-free CYP46A1 is in orange, cholesterol sulfate-bound is in cyan, cholesterol sulfate is in yellow, and heme is in pink. The 230–239 region is disordered in substrate-free CYP46A1 and is therefore not shown.

Figure 4

Peptides showing altered H-D exchange upon binding of cholesterol. The scale of the y axis shows the expected number of deuterated amide groups in each peptide. Adjustment for the back-exchange was not performed; therefore, the difference in deuterium incorporation between substrate-free (○) and cholesterol-bound (■) CYP46A1 is a relative value.

Figure 5

Peptides showing altered H-D exchange upon binding of 24S-hydroxycholesterol. The scale of the y axis shows the expected number of deuterated amide groups in each peptide. Adjustment for the back-exchange was not performed; therefore, the difference in deuterium incorporation between substrate-free (○) and 24S-hydroxycholesterol-bound (■) CYP46A1 is a relative value.

Figure 6

Peptides showing altered H-D exchange upon binding of Adx to substrate-free and cholesterol sulfate-bound CYP46A1. The 225–239 peptide in cholesterol sulfate-bound CYP46A1 is shown for comparison. The scale of the y axis shows the expected number of deuterated amide groups in each peptide. Adjustment for the back-exchange was not performed; therefore, the difference in deuterium incorporation in the absence (○) and presence (■) of Adx is a relative value.

Figure 7

Regions in substrate-free CYP46A1 exhibiting altered deuterium incorporation upon binding of cholesterol (in red). Corresponding peptides in cholesterol sulfate-bound are in cyan. Unaffected regions in substrate-free CYP46A1 are in orange. Cholesterol sulfate is in yellow and heme is in pink. F121, V126, R147, N227, A367, and K358 are in blue. The shape of the active site in substrate-free CYP46A1 is shown in orange mesh and in cholesterol-sulfate-bound P450 is in cyan mesh.

Figure 8

Regions in substrate-free CYP46A1 exhibiting altered deuterium incorporation upon binding of 24S-hydroxycholesterol (in light blue). The unaffected 351–361 peptide is in orange. Heme is in pink, A367 and F121 are in blue, and the shape of the active site in substrate-free CYP46A1 is in olive mesh.

Figure 9

Regions in CYP46A1 affected by the binding of Adx. Peptides in substrate-free CYP46A1 are shown in orange, and in cholesterol sulfate-bound P450 are in cyan. Cholesterol sulfate is in yellow, heme is in pink, R147, N227, I301, A302, T306, and K358 are in blue. The 230–239 region is disordered in substrate-free CYP46A1 and is therefore not shown.