Transporter proteins from the multidrug and toxic compound extrusion (MATE)1 family play vital roles in metabolite transport in plants2-3, directly affecting crop yields worldwide4. MATE transporters also mediate multidrug resistance (MDR) in bacteria and mammals5, modulating the efficacy of many pharmaceutical drugs used in the treatment of a variety of diseases6-9. MATE transporters couple substrate transport to electrochemical gradients and are the only remaining class of MDR transporters whose structure has not been determined10. Here we report the x-ray structure of the MATE transporter NorM from Vibrio cholerae determined to 3.65 Å, revealing an outward-facing conformation with two portals open to the outer leaflet of the membrane and a unique topology of the predicted 12 transmembrane helices distinct from any other known MDR transporter. We also report a cation-binding site in close proximity to residues previously deemed critical for transport11. This conformation likely represents a stage of the transport cycle with high-affinity to monovalent cations and low-affinity to substrates.

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).

General procedure. NMR spectra were recorded on Bruker DRX-500, AMX-500 or AMX-300 instruments. IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR spectrometer. High-resolution mass spectra (HRMS) were recorded on a VG ZAB-ZSE mass spectrometer using MALDI (matrix-assisted laser-desorption ionization) or ESI (electrospray ionization).3α,7α,12α,24-Tetrahydroxycholane (3): [1] To a suspension of LiAlH 4 (3.85 g, 100 mmol) in dry THF (100 mL) at 0°C was added dropwise a solution of cholic acid (15.03 g, 35 mmol) in dry THF (200 mL) with vigorous stirring under nitrogen atmosphere. The reaction mixture was then heated to reflux with stirring for overnight. Upon completion, the reaction was carefully quenched with saturated aqueous NH 4 Cl solution at RT. Then the mixture was acidified with 1N HCl to pH 1~2. The precipitate was collected via filtration and washed with water and acetone to give the product 2 (12.1 g, 88%) as a white solid.7α,12α-Dihydroxycholane (2): To a solution of tetrahydroxycholane 3 (17.4 g, 44.2 mmol) and triethylamine (11.8 g, 116.6 mmol) in dry THF (300 mL) was added dropwise a solution of methanesulfonyl chloride (11.1 g, 97 .2 mmol) in dry THF (100 mL) at 0°C. Then the reaction mixture was slowly warmed up to RT. After 30 minutes, the reaction was quenched with saturated aqueous NH 4 Cl solution. The organic solvents were removed under vacuum and the left aqueous solution was extracted with ethyl acetate. The combined organic portions were washed with brine and then dried over anhydrous Na 2 SO 4 . The filtered solution was concentrated under vacuum to give the product 7α,12α-Dihydro-3α,24-dimethylsulfonate-cholane (25.6 g, 95%) which was directly dissolved in dry THF for the next step.A solution of LiAlH 4 (6.0 g, 158 mmol) in dry THF (300 mL) was added dropwise to the above obtained THF solution of 7α,12α-Dihydro-3α,24-dimethylsulfonate-cholane at 0°C. The reaction mixture was heated to reflux with stirring for overnight. Then the reaction was quenched with saturated aqueous NH 4 Cl solution at RT. The organic solvents were evaporated under vacuum and the left aqueous solution was acidified with 1N HCl to pH 1. The white precipitate formed was collected via filtration, washed with water and acetone to give the crude product, and then crystallized in dichloromethane and methanol to afford the pure compound 7α,12α-dihydrocholane (12.8 g, 80% over two steps 7α, 12α-Di-(O-β-D-maltosyl)-cholane (1):A mixture of compound 2 (210 mg, 0.58 mmol), 1-thio-ethyl-hepta-o-benzoyl-β-D-maltose (2.1 g, 2.08 mmol) and 4 Ǻ molecule sieves (800 mg) in dry CH 2 Cl 2 (50 mL) was stirred at RT for 30 minutes. The reaction mixture was then cooled to -15 °C, to which was added crystallized N-iodosuccinimide (500 mg, 2.22 mmol) and silver trifluorosulfonate (100 mg, 0.39 mmol). The reaction mixture was slowly warmed up to RT with stirring. The reaction was monitored by TLC. Upon completion, the reaction was quenched with triethylamine. The mixture was filtered and the filtrate was concentrated under vac...

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-Å resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.Cytochromes P450 (P450s) belong to a superfamily of heme-containing monooxygenases and are the predominant enzyme responsible for phase I metabolism of clinically relevant drugs (Wang and Tompkins, 2008). Through the incorporation of a single oxygen atom, P450s generate products that are more water-soluble and are either readily excreted in the urine or more amenable substrates for phase II conjugation. Previous studies have demonstrated that many of these enzymes are highly flexible (Domanski and Halpert, 2001;, allowing them to accommodate a wide range of substrates, including numerous steroids, pharmaceuticals, and environmental pollutants (Johnson and Stout, 2005). P450 2B enzymes were among the first mammalian P450s to be purified and cloned and have served as a prototype for biochemical and biophysical experiments, as well as studies of substrate specificity and of interactions with the redox

Amphiphile selection is a critical step for structural studies of membrane proteins (MPs). We have developed a family of steroidbased 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 seventransmembrane 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.

Prior X-ray crystal structures of rabbit cytochrome P450 2B4 (2B4) in complex with various imidazoles have demonstrated markedly different enzyme conformations depending on the size of the inhibitor occupying the active site. In this study, structures of 2B4 were solved with the antiplatelet drugs clopidogrel and ticlopidine, which were expected to have greater freedom of movement in the binding pocket. Ticlopidine could be modeled into the electron density maps in two distinct orientations, both of which are consistent with metabolic data gathered with other mammalian P450 enzymes. Results of ligand docking and heme-induced NMR relaxation of drug protons showed that ticlopidine was preferentially oriented with the chlorophenyl group closest to the heme. Because of its stereocenter, clopidogrel was easier to fit in the electron density and exhibited a single orientation, which points the chlorophenyl ring toward the heme. The Cα traces of both complexes aligned very well to each other and revealed a compact, closed structure that resembles the conformation observed in two previously solved 2B4 structures with the small molecule inhibitors 4-(4-chlorophenyl)imidazole and 1-(4-chlorophenyl)imidazole. The 2B4 active site is able to accommodate small ligands by moving only a small number of side chains, suggesting that ligand reorientation is energetically favored over protein conformational changes for binding of these similar sized molecules. Adjusting both protein conformation and ligand orientation in the active site gives 2B4 the flexibility to bind to the widest range of molecules, while also being energetically favorable.

A challenging requirement for X-ray crystallography or NMR structure determination of membrane proteins (MPs), in contrast to soluble proteins, is the necessary use of amphiphiles to mimic the hydrophobic environment of membranes. A number of new detergents, lipids and non-detergent-like amphiphiles have been developed that stabilize MPs, and these have contributed to increased success in MP structural determinations in recent years. Despite some successes, currently available reagents are inadequate and there remains a pressing need for new amphiphiles. Literature examples and some new developments are selected here as a framework for discussing desirable properties of new amphiphiles for MP structural biology.

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized beta-barrel E. coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with microgram amounts of uniformly (2)H, (15)N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [ (15)N, (1)H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX.