C.D. Stout

The Structure of Human Cytochrome P450 2C9 Complexed with Flurbiprofen at 2.0 A Resolution

The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 Å by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate for regioselective oxidation in a relatively large active site cavity and are likely to account for the high catalytic efficiency exhibited by P450 2C9 for the regioselective oxidation of several anionic non-steroidal anti-inflammatory drugs. The structure provides a basis for interpretation of a number of observations regarding the substrate selectivity of P450 2C9 and the observed effects of mutations on catalysis.

Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2.

Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor α-naphthoflavone, determined to a resolution of 1.95 Å. α-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members.

Refined crystal structure of Cd, Zn metallothionein at 2.0Åresolution

The crystal structure of Cd5,Zn2-metallothionein from rat liver has been refined at 2.0Aresolution of a R-value of 0.176 for all observed data. The five Cd positions in the asymmetric unit of the crystal create a pseudo-centrosymmetric constellation about a crystallographic 2-fold axis. Consequently, the distribution of anomalous differences is almost ideally centrosymmetric. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calculate a new electron density map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calculated from this model predict the positions of three sites in a (NH4)2WS4 derivative. Single isomorphous replacement phases calculated with these tungsten sites confirm the positions of the Cd sites from the new direct methods calculations. The refined metallothionein structure has a root-mean-square deviation of 0.016Afrom ideality of bonds and normal stereochemistry of φ, ϑ and χ torsion angles. The metallothionein crystal structure is in agreement with the structures for the α and β domains in solution derived by nuclear magnetic resonance methods. The overall chain folds and all metal to cysteine bonds are the same in the two structure determinations. The handedness of a short helix in the α-domain (residues 41 to 45) is the same in both structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochemistry. Short cysteine peptide sequences repeated in the structure adopt restricted conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate association of molecules about the diagonal 2-fold axes and trapped ions of crystallization (modeled as phosphate and sodium). Variation in the chemical and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins.

Structures of human microsomal cytochrome P450 2A6 complexed with coumarin and methoxsalen

Human microsomal cytochrome P450 2A6 (CYP2A6) contributes extensively to nicotine detoxication but also activates tobacco-specific procarcinogens to mutagenic products. The CYP2A6 structure shows a compact, hydrophobic active site with one hydrogen bond donor, Asn297, that orients coumarin for regioselective oxidation. The inhibitor methoxsalen effectively fills the active site cavity without substantially perturbing the structure. The structure should aid the design of inhibitors to reduce smoking and tobacco-related cancers.

Crystal structure of an 82-nucleotide RNA-DNA complex formed by the 10-23 DNA enzyme.

The structure of a large nucleic acid complex formed by the 10–23 DNA enzyme bound to an RNA substrate was determined by X–ray diffraction at 3.0 Å resolution. The 82–nucleotide complex contains two strands of DNA and two strands of RNA that form five double–helical domains. The spatial arrangement of these helices is maintained by two four–way junctions that exhibit extensive base–stacking interactions and sharp turns of the phosphodiester backbone stabilized by metal ions coordinated to nucleotides at these junctions. Although it is unlikely that the structure corresponds to the catalytically active conformation of the enzyme, it represents a novel nucleic acid fold with implications for the Holliday junction structure.

Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution.

The crystal structure of Cd5,Zn2-metallothionein from rat liver has been refined at 2.0 A resolution of a R-value of 0.176 for all observed data. The five Cd positions in the asymmetric unit of the crystal create a pseudo-centrosymmetric constellation about a crystallographic 2-fold axis. Consequently, the distribution of anomalous differences is almost ideally centrosymmetric. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calculate a new electron density map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calculated from this model predict the positions of three sites in a (NH4)2WS4 derivative. Single isomorphous replacement phases calculated with these tungsten sites confirm the positions of the Cd sites from the new direct methods calculations. The refined metallothionein structure has a root-mean-square deviation of 0.016 A from ideality of bonds and normal stereochemistry of phi, phi and chi torsion angles. The metallothionein crystal structure is in agreement with the structures for the alpha and beta domains in solution derived by nuclear magnetic resonance methods. The overall chain folds and all metal to cysteine bonds are the same in the two structure determinations. The handedness of a short helix in the alpha-domain (residues 41 to 45) is the same in both structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochemistry. Short cysteine peptide sequences repeated in the structure adopt restricted conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate association of molecules about the diagonal 2-fold axes and trapped ions of crystallization (modeled as phosphate and sodium). Variation in the chemical and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins.

Determinants of cytochrome P450 2C8 substrate binding: structures of complexes with montelukast, troglitazone, felodipine, and 9-cis-retinoic acid.

Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 Å), troglitazone (2.7 Å), felodipine (2.3 Å), and 9-cis-retinoic acid (2.6 Å) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates.

Atomically defined mechanism for proton transfer to a buried redox centre in a protein

The basis of the chemiosmotic theory is that energy from light or respiration is used to generate a trans-membrane proton gradient. This is largely achieved by membrane-spanning enzymes known as ‘proton pumps’. There is intense interest in experiments which reveal, at the molecular level, how protons are drawn through proteins.Here we report the mechanism, at atomic resolution, for a single long-range electron-coupled proton transfer. In Azotobacter vinelandii ferredoxin I, reduction of a buried iron–sulphur cluster draws in a solvent proton, whereas re-oxidation is ‘gated’ by proton release to the solvent. Studies of this ‘proton-transferring module’ by fast-scan protein film voltammetry, high-resolution crystallography, site-directed mutagenesis and molecular dynamics, reveal that proton transfer is exquisitely sensitive to the position and pK of a single amino acid. The proton is delivered through the protein matrix by rapid penetrative excursions of the side-chain carboxylate of a surface residue (Asp 15), whose pK shifts in response to the electrostatic charge on the iron–sulphur cluster. Our analysis defines the structural, dynamic and energetic requirements for proton courier groups in redox-driven proton-pumping enzymes.

Refinement of the 7 Fe ferredoxin from Azotobacter vinelandii at 1.9 A resolution.

The recently redetermined structure of the 7 Fe ferredoxin from Azotobacter vinelandii has been refined against a new 1.9 A data set. The crystallographic R-factor is 0.215 for all 9586 observed reflections 8.0 to 1.9 A. The model contains 106 amino acid residues, two Fe-S clusters and 21 water molecules. The root-mean-square deviations from ideality of bonds and angles are 0.014 A and 3.3 degrees, respectively. The refinement confirms the presence of two free cysteines: the thiol of C11 is in association with the side-chain of K100; the thiol of C24 is 3.35 A from inorganic sulfur of the [4 Fe-4 S] cluster. The refinement confirms a [3 Fe-4 S] model for the 3 Fe cluster. The two Fe-S clusters have similar bond distances and angles. The structure of the protein for residues 1 to 57 superposes within 0.85 A on residues 1 to 53 of the 8 Fe ferredoxin structure for main-chain N, CA and C atoms, if residues 9, 10, 29 and 30 of 7 Fe ferredoxin are omitted. These residues are part of two loops in contact with residues of the extended C-terminal chain of 7 Fe ferredoxin.

Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins.

Significance Membrane proteins (MPs) perform a variety of essential cellular functions, account for about one-third of encoded proteins in genomes, and comprise more than one-half of human drug targets. High-resolution structures are essential to understand the underlying molecular mechanisms of MPs and facilitate structure-based drug design efforts. Detergents are indispensible in the solubilization of MPs, but they tend to destabilize MPs and often impede the growth of well-ordered protein crystals. We describe a class of structurally unique detergents, designated as facial amphiphiles, which improved MP stability and success in the crystallization of different families of MPs. Amphiphile selection is a critical step for structural studies of membrane proteins (MPs). We have developed a family of steroid-based facial amphiphiles (FAs) that are structurally distinct from conventional detergents and previously developed FAs. The unique FAs stabilize MPs and form relatively small protein–detergent complexes (PDCs), a property considered favorable for MP crystallization. We attempted to crystallize several MPs belonging to different protein families, including the human gap junction channel protein connexin 26, the ATP binding cassette transporter MsbA, the seven-transmembrane G protein-coupled receptor-like bacteriorhodopsin, and cytochrome P450s (peripheral MPs). Using FAs alone or mixed with other detergents or lipids, we obtained 3D crystals of the above proteins suitable for X-ray crystallographic analysis. The fact that FAs enhance MP crystallizability compared with traditional detergents can be attributed to several properties, including increased protein stability, formation of small PDCs, decreased PDC surface flexibility, and potential to mediate crystal lattice contacts.