The structure of P450 3A4 was determined by x-ray crystallography to 2.05-Å resolution. P450 3A4 catalyzes the metabolic clearance of a large number of clinically used drugs, and a number of adverse drug-drug interactions reflect the inhibition or induction of the enzyme. P450 3A4 exhibits a relatively large substrate-binding cavity that is consistent with its capacity to oxidize bulky substrates such as cyclosporin, statins, taxanes, and macrolide antibiotics. Family 3A P450s also exhibit unusual kinetic characteristics that suggest simultaneous occupancy by smaller substrates. Although the active site volume is similar to that of P450 2C8 (PDB code: 1PQ2), the shape of the active site cavity differs considerably due to differences in the folding and packing of portions of the protein that form the cavity. Compared with P450 2C8, the active site cavity of 3A4 is much larger near the heme iron. The lower constraints on the motions of small substrates near the site of oxygen activation may diminish the efficiency of substrate oxidation, which may, in turn, be improved by space restrictions imposed by the presence of a second substrate molecule. The structure of P450 3A4 should facilitate a better understanding of the substrate selectivity of the enzyme.Determination of the structure of P450 1 3A4 is of particular interest because the enzyme contributes extensively to human drug metabolism due to its high level of expression in liver (1) and broad capacity to oxidize structurally diverse substrates (2, 3). The enzyme also provides a significant barrier to the bioavailability of new drug candidates contributing to attrition from the developmental pipeline. Additionally, metabolic drug-drug interactions between substrates and inhibitors of the enzyme can profoundly affect the safety or efficacy of drug therapy (4, 5).Our laboratory was the first to demonstrate that microsomal P450s could be crystallized for structural determination by x-ray crystallography when the proteins were modified for expression as conditional membrane proteins (6, 7). As a result, structures for P450s in family 2, subfamilies B and C are now available (8 -14). P450s of family 3, subfamily A exhibit less than 40% amino acid sequence identity with family 2 P450s. In addition, family 3 P450s often exhibit complex kinetic properties such as substrate and effector activation. Effectors or alternative substrates can modulate the apparent binding affinity for other inhibitors (15) and substrates (16). Moreover, there are a number of examples where alternative substrates fail to inhibit the oxidation of specific substrates leading to kinetic models based on the occupancy of the substrate-binding cavity by two substrates that each can be oxidized by the reactive, hypervalent oxy-perferryl heme intermediate without interference from the other (17, 18). The observation that P450 3A4 oxidizes some of the largest substrates identified for P450s, such as cyclosporin, bromocryptine, and macrolide antibiotics (3), has generally suggested the likelihood that...

A 1.9-Å molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B and C, the N terminus of helix I, and the ␤ 4 region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F and G helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding. Cytochromes P450 (P450)1 are involved in steroidogenesis, fatty acid metabolism, synthesis of bile and retinoid acids, and production of plant toxins, but it is their function in the elimination of xenobiotics that has received the most attention. In mammals, xenobiotic metabolizing P450s play the central role in detoxification of hydrophobic drugs, carcinogens, and toxins by decreasing the lipid solubility of these chemicals and, thus, promoting excretion. In contrast to the strict substrate selectivity of classical enzymes, xenobiotic-metabolizing P450s can each bind and oxidize a set of substrates with distinct sizes, shapes, and stereochemical features. Although the variety of substrates binding to a given P450 is often broad, the oxidation of each is usually remarkably regiospecific and stereospecific.Identification of the structural basis for the specific monooxygenation and binding of a diverse but select set of substrates has been a particularly challenging goal, which is an important prerequisite for understanding selective substrate oxidation. Although the diversity of substrates might suggest an easily accessible active site, initial structures of both soluble bacterial (1) and microsomal mammalian (2) P450s revealed active sites buried within the globular structure of the protein.A few recent bacterial structures, however, suggest a "lid" domain composed of helices F and G, the motion of which controls substrate entry (3-5). Protein ...

Cytochrome P450 (CYP) 24A1 catalyzes the side-chain oxidation of the hormonal form of vitamin D. Expression of CYP24A1 is up-regulated to attenuate vitamin-D signaling associated with calcium homeostasis and cellular growth processes. The development of therapeutics for disorders linked to vitamin D-insufficiency would be greatly facilitated by structural knowledge of CYP24A1. Here we report the crystal structure of rat CYP24A1 at 2.5 Å resolution. The structure exhibits an open cleft leading to the active site heme prosthetic group on the distal surface that is likely to define the path of substrate access into the active site. The entrance to the cleft is flanked by conserved hydrophobic residues on helices A′ and G′ suggesting a mode of insertion into the inner mitochondrial membrane. A docking model for 1α,25-(OH) 2 D 3 binding in the open form of CYP24A1 is proposed that clarifies the structural determinants of secosteroid recognition and validates the predictive power of existing homology models of CYP24A1. Analysis of CYP24A1's proximal surface identifies the determinants of adrenodoxin recognition as a constellation of conserved residues from helices K, K″ and L that converge with an adjacent lysine-rich loop for binding the redox protein. Overall, the CYP24A1 structure provides the first template for understanding membrane insertion, substrate binding, and redox partner interaction in mitochondrial P450s. Data Deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3K9V and 3K9Y).

The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.

An open-ended hollow tubular structure is designed based on hydrogen-bond-directed self-assembly of a chimeric cyclic peptide subunit comprised of alternating alpha- and epsilon-amino acids. The design features a novel 1,4-disubstituted-1,2,3-triazole epsilon-amino acid and its utility as a peptide backbone substitute. The N-Fmoc-protected epsilon-amino acid was synthesized in high yield and optical purity in three steps from readily available starting materials and was employed in solid-phase peptide synthesis to afford the desired cyclic peptide structure. The cyclic peptide self-assembly has been studied in solution by (1)H NMR and mass spectrometry and the resulting tubular ensemble characterized in the solid state by X-ray crystallography.