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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.

SUBMITTER: White MA

PROVIDER: S-EPMC2467524 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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

White Mark Andrew MA Mast Natalia N Bjorkhem Ingemar I Bjorkhem Ingemar I Johnson Eric F EF Stout C David CD Pikuleva Irina A IA

Acta crystallographica. Section D, Biological crystallography 20080419 Pt 5

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 compleme ...[more]

PMID: 18453684

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Project description:Cytochrome P450 46A1 encoded by CYP46A1 catalyzes cholesterol 24-hydroxylation and is a CNS-specific enzyme that controls cholesterol removal and turnover in the brain. Accumulating data suggest that increases in cytochrome P450 46A1 activity in mouse models of common neurodegenerative diseases affect various, apparently unlinked biological processes and pathways. Yet, the underlying reason for these multiple enzyme activity effects is currently unknown. Herein, we tested the hypothesis that cytochrome P450 46A1-mediated sterol flux alters physico-chemical properties of the plasma membranes and thereby membrane-dependent events. We used 9-month old 5XFAD mice (an Alzheimer's disease model) treated for 6 months with the anti-HIV drug efavirenz. These animals have previously been shown to have improved behavioral performance, increased cytochrome P450 46A1 activity in the brain, and increased sterol flux through the plasma membranes. We further examined 9-month old Cyp46a1-/- mice, which have previously been observed to have cognitive deficits and decreased sterol flux through brain membranes. Synaptosomal fractions from the brain of efavirenz-treated 5XFAD mice had essentially unchanged cholesterol levels as compared to control 5XFAD mice. However with efavirenz treatment in these mice, there were changes in the membrane properties (increased cholesterol accessibility, ordering, osmotic resistance, and thickness) as well as total glutamate content and ability to release glutamate in response to mild stimulation. Similarly, the cholesterol content in synaptosomal fractions from the brain of Cyp46a1-/- mice was essentially the same as in wild type mice but knockout of Cyp46a1 was associated with changes in membrane properties and glutamate content and its exocytotic release. Changes in Cyp46a1-/- mice were in the opposite direction to those observed in efavirenz-treated vs control 5XFAD mice. Incubation of synaptosomal fractions with the inhibitors of glycogen synthase kinase 3, cyclin-dependent kinase 5, protein phosphatase 1/2A or calcineurin, and protein phosphatase 2B revealed that increased sterol flux in efavirenz-treated vs control 5XFAD mice affected the ability of all four enzymes to modulate glutamate release. In contrast, in Cyp46a1-/-vs wild type mice, decreased sterol flux altered the ability of only cyclin-dependent kinase 5 and protein phosphatase 2B to regulate the glutamate release. Collectively, our results support cytochrome P450 46A1-mediated sterol flux as an important contributor to the fundamental properties of the membranes, protein phosphorylation, and synaptic transmission Also, our data provide an explanation of how one enzyme, cytochrome P450 46A1, can affect multiple pathways and processes and serve as a common potential target for several neurodegenerative disorders.

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

Publications

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

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