Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase.

The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton-motive force to drive hydride transfer from NADH to NADP+ in bacteria and eukaryotes. Here we solved a 2.2-Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular dynamics simulations, we show that transient water flows across a narrow pore and a hydrophobic "dry" region in the middle of the membrane channel, with key residues His42α2 (chain A) being protonated and Thr214ß (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214ß to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.

Halogen-π Interactions in the Cytochrome P450 Active Site: Structural Insights into Human CYP2B6 Substrate Selectivity.

Numerous cytochrome P450 (CYP) 2B6 substrates including drugs and environmental chemicals are halogenated. To assess the role of halogen-π bonds in substrate selectivity and orientation in the active site, structures of four CYP2B6 monoterpenoid complexes were solved by X-ray crystallography. Bornyl bromide exhibited dual orientations in the active site with the predominant orientation revealing a bromine-π bond with the Phe108 side chain. Bornane demonstrated two orientations with equal occupancy; in both, the C2 atom that bears the bromine in bornyl bromide was displaced by more than 2.5 Å compared with the latter complex. The bromine in myrtenyl bromide π-bonded with Phe297 in CYP2B6, whereas the two major orientations in the active site mutant I114V exhibited bromine-π interactions with two additional residues, Phe108 and Phe115. Analysis of existing structures suggests that halogen-π interactions may be unique to the CYP2B enzymes within CYP family 2 but are also important for CYP3A enzymes.

Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase.

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O-O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O-O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion of O2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba3.

Effect of detergent binding on cytochrome P450 2B4 structure as analyzed by X-ray crystallography and deuterium-exchange mass spectrometry.

Multiple crystal structures of CYP2B4 have demonstrated the binding of the detergent 5-cyclohexyl-1-pentyl-ß-D-maltoside (CYMAL-5) in a peripheral pocket located adjacent to the active site. To explore the consequences of detergent binding, X-ray crystal structures of the peripheral pocket mutant CYP2B4 F202W were solved in the presence of hexaethylene glycol monooctyl ether (C8E6) and CYMAL-5. The structure in the presence of CYMAL-5 illustrated a closed conformation indistinguishable from the previously solved wild-type. In contrast, the F202W structure in the presence of C8E6 revealed a detergent molecule that coordinated the heme-iron and extended to the protein surface through the substrate access channel 2f. Despite the overall structural similarity of these detergent complexes, remarkable differences were observed in the A, A', and H helices, the F-G cassette, the C-D and ß4 loop region. Hydrogen-deuterium exchange mass spectrometry (DXMS) was employed to probe these differences and to test the effect of detergents in solution. The presence of either detergent increased the H/D exchange rate across the plastic regions, and the results obtained by DXMS in solution were consistent in general with the relevant structural snapshots. The study provides insight into effect of detergent binding and the interpretation of associated conformational dynamics of CYP2B4.

Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations.

Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH3, or -CF3 at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-α-pinene was solved at 2.2 Å and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 Å with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.

Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism.

Crystal structures of CYP2B35 and CYP2B37 from the desert woodrat were solved in complex with 4-(4-chlorophenyl)imidazole (4-CPI). The closed conformation of CYP2B35 contained two molecules of 4-CPI within the active site, whereas the CYP2B37 structure demonstrated an open conformation with three 4-CPI molecules, one within the active site and the other two in the substrate access channel. To probe structure-function relationships of CYP2B35, CYP2B37, and the related CYP2B36, we tested the O-dealkylation of three series of related substrates-namely, 7-alkoxycoumarins, 7-alkoxy-4-(trifluoromethyl)coumarins, and 7-alkoxy-4-methylcoumarins-with a C1-C7 side chain. CYP2B35 showed the highest catalytic efficiency (kcat/KM) with 7-heptoxycoumarin as a substrate, followed by 7-hexoxycoumarin. In contrast, CYP2B37 showed the highest catalytic efficiency with 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), followed by 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC). CYP2B35 had no dealkylation activity with 7-MFC or 7-EFC. Furthermore, the new CYP2B-4-CPI-bound structures were used as templates for docking the 7-substituted coumarin derivatives, which revealed orientations consistent with the functional studies. In addition, the observation of multiple -Cl and -NH-π interactions of 4-CPI with the aromatic side chains in the CYP2B35 and CYP2B37 structures provides insight into the influence of such functional groups on CYP2B ligand binding affinity and specificity. To conclude, structural, computational, and functional analysis revealed striking differences between the active sites of CYP2B35 and CYP2B37 that will aid in the elucidation of new structure-activity relationships.

Contributions of ionic interactions and protein dynamics to cytochrome P450 2D6 (CYP2D6) substrate and inhibitor binding.

P450 2D6 contributes significantly to the metabolism of >15% of the 200 most marketed drugs. Open and closed crystal structures of P450 2D6 thioridazine complexes were obtained using different crystallization conditions. The protonated piperidine moiety of thioridazine forms a charge-stabilized hydrogen bond with Asp-301 in the active sites of both complexes. The more open conformation exhibits a second molecule of thioridazine bound in an expanded substrate access channel antechamber with its piperidine moiety forming a charge-stabilized hydrogen bond with Glu-222. Incubation of the crystalline open thioridazine complex with alternative ligands, prinomastat, quinidine, quinine, or ajmalicine, displaced both thioridazines. Quinine and ajmalicine formed charge-stabilized hydrogen bonds with Glu-216, whereas the protonated nitrogen of quinidine is equidistant from Asp-301 and Glu-216 with protonated nitrogen H-bonded to a water molecule in the access channel. Prinomastat is not ionized. Adaptations of active site side-chain rotamers and polypeptide conformations were evident between the complexes, with the binding of ajmalicine eliciting a closure of the open structure reflecting in part the inward movement of Glu-216 to form a hydrogen bond with ajmalicine as well as sparse lattice restraints that would hinder adaptations. These results indicate that P450 2D6 exhibits sufficient elasticity within the crystal lattice to allow the passage of compounds between the active site and bulk solvent and to adopt a more closed form that adapts for binding alternative ligands with different degrees of closure. These crystals provide a means to characterize substrate and inhibitor binding to the enzyme after replacement of thioridazine with alternative compounds.

Structural and biophysical characterization of human cytochromes P450 2B6 and 2A6 bound to volatile hydrocarbons: analysis and comparison.

X-ray crystal structures of complexes of cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity. Furthermore, results from isothermal titration calorimetry indicated a much more substantial contribution of favorable enthalpy to sabinene binding to CYP2B6 as opposed to CYP2A6, consistent with the previous observations with (+)-α-pinene. Structural analysis of CYP2B6 complexes with sabinene and the structurally similar (3)-carene and comparison with previously solved structures revealed how the movement of the F206 side chain influences the volume of the binding pocket. In addition, retrospective analysis of prior structures revealed that ligands containing -Cl and -NH functional groups adopted a distinct orientation in the CYP2B active site compared with other ligands. This binding mode may reflect the formation of Cl-π or NH-π bonds with aromatic rings in the active site, which serve as important contributors to protein-ligand binding affinity and specificity. Overall, the findings from multiple techniques illustrate how drugs metabolizing CYP2B6 and CYP2A6 handle a common hydrocarbon found in the environment. The study also provides insight into the role of specific functional groups of the ligand that may influence the binding to CYP2B6.

Towards novel therapeutics for HIV through fragment-based screening and drug design.

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-µM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

Conserved glycine 232 in the ligand channel of ba3 cytochrome oxidase from Thermus thermophilus.

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617-11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Structural and thermodynamic basis of (+)-α-pinene binding to human cytochrome P450 2B6.

Despite recent advances in atomic-level understanding of drug and inhibitor interactions with human cytochromes P450, the decades-old questions of chemical and structural determinants of hydrocarbon binding are still unanswered. (+)-α-Pinene is a monoterpene hydrocarbon that is widely distributed in the environment and a potent P450 2B inhibitor. Therefore, a combined biophysical and structural analysis of human P450 2B6 interactions with (+)-α-pinene was undertaken to elucidate the basis of the very high affinity binding. Binding of (+)-α-pinene to the P450 active site was demonstrated by a Type I spectral shift. Thermodynamics of ligand binding were explored using isothermal titration calorimetry and compared to those of P450 2A6, which is much less flexible than 2B6 based on comparison of multiple X-ray crystal structures. Consistent with expectation, entropy is the major driving force for hydrocarbon binding to P450 2A6, as evidenced by the calorimetric results. However, formation of the 2B6-(+)-α-pinene complex has a significant enthalpic component. A 2.0 Å resolution crystal structure of this enzyme-ligand complex reveals that the highly plastic 2B6 utilizes previously unrecognized rearrangements of protein motifs. The results indicate that the specific components of enthalpic contribution to ligand binding are closely tied to the degree of enzyme flexibility.

Structural diversity of eukaryotic membrane cytochrome p450s.

X-ray crystal structures are available for 29 eukaryotic microsomal, chloroplast, or mitochondrial cytochrome P450s, including two non-monooxygenase P450s. These structures provide a basis for understanding structure-function relations that underlie their distinct catalytic activities. Moreover, structural plasticity has been characterized for individual P450s that aids in understanding substrate binding in P450s that mediate drug clearance.

AutoDrug: fully automated macromolecular crystallography workflows for fragment-based drug discovery.

AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallography steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully demonstrated. This workflow was run once on the same 96 samples that the group had examined manually and the workflow cycled successfully through all of the samples, collected data from the same samples that were selected manually and located the same peaks of unmodeled density in the resulting difference Fourier maps.

A structural snapshot of CYP2B4 in complex with paroxetine provides insights into ligand binding and clusters of conformational states.

An X-ray crystal structure of CYP2B4 in complex with the drug paroxetine [(3S,4R)-3-[(2H-1,3-benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine] was solved at 2.14 Å resolution. The structure revealed a conformation intermediate to that of the recently solved complex with amlodipine and that of the more compact complex with 4-(4-chlorophenyl)imidazole in terms of the placement of the F-G cassette. Moreover, comparison of the new structure with 15 previously solved structures of CYP2B4 revealed some new insights into the determinants of active-site size and shape. The 2B4-paroxetine structure is nearly superimposable on a previously solved closed structure in a ligand-free state. Despite the overall conformational similarity among multiple closed structures, the active-site cavity volume of the paroxetine complex is enlarged. Further analysis of the accessible space and binding pocket near the heme reveals a new subchamber that resulted from the movement of secondary structural elements and rearrangements of active-site side chains. Overall, the results from the comparison of all 16 structures of CYP2B4 demonstrate a cluster of protein conformations that were observed in the presence or absence of various ligands.

Antifungal Azoles: Structural Insights into Undesired Tight Binding to Cholesterol-Metabolizing CYP46A1.

Although there are currently three generations of antifungal azoles on the market, even the third-generation agents show undesirable interactions with human cytochrome P450 (P450) enzymes. CYP46A1 is a cholesterol-metabolizing P450 in the brain that tightly binds a number of structurally distinct azoles. Previously, we determined the crystal structures of CYP46A1 in complex with voriconazole and clotrimazole, and in the present work we cocrystallized the P450 with posaconazole at 2.5 Å resolution. This long antifungal drug coordinates the P450 heme iron with the nitrogen atom of its terminal azole ring and adopts a linear configuration occupying the whole length of the substrate access channel and extending beyond the protein surface. Numerous drug-protein interactions determine the submicromolar Kd of posaconazole for CYP46A1. We compared the crystal structure of posaconazole-bound CYP46A1 with those of the P450 in complex with other drugs, including the antifungal voriconazole and clotrimazole. We also analyzed the accommodation of posaconazole in the active site of the target enzymes, CYPs 51, from several pathogenic species. These and the solution studies with different marketed azoles, collectively, allowed us to identify the determinants of tight azole binding to CYP46A1 and generate an overall picture of azole binding to this important P450. The data obtained suggest that development of CYP51-specific antifungal agents will continue to be a challenge. Therefore, structural understanding of the azole binding not only to CYPs 51 from the pathogenic species but also to different human P450s is required to deal efficiently with this challenge.

Small molecule regulation of protein conformation by binding in the Flap of HIV protease.

The fragment indole-6-carboxylic acid (1F1), previously identified as a flap site binder in a fragment-based screen against HIV protease (PR), has been cocrystallized with pepstatin-inhibited PR and with apo-PR. Another fragment, 3-indolepropionic acid (1F1-N), predicted by AutoDock calculations and confirmed in a novel inhibition of nucleation crystallization assay, exploits the same interactions in the flap site in two crystal structures. Both 1F1 and 1F1-N bind to the closed form of apo-PR and to pepstatin:PR. In solution, 1F1 and 1F1-N raise the Tm of apo-PR by 3.5-5 °C as assayed by differential scanning fluorimetry (DSF) and show equivalent low-micromolar binding constants to both apo-PR and pepstatin:PR, assayed by backscattering interferometry (BSI). The observed signal intensities in BSI are greater for each fragment upon binding to apo-PR than to pepstatin-bound PR, consistent with greater conformational change in the former binding event. Together, these data indicate that fragment binding in the flap site favors a closed conformation of HIV PR.

Binding of a cyano- and fluoro-containing drug bicalutamide to cytochrome P450 46A1: unusual features and spectral response.

Cytochrome P450 46A1 (CYP46A1) is the cholesterol 24-hydroxylase initiating the major pathways of cholesterol removal from the brain, and bicalutamide (BIC) is a drug of choice for the treatment of progressive androgen-dependent prostate cancer. We evaluated the interactions of BIC with CYP46A1 by x-ray crystallography and by conducting solution and mutagenesis studies. Because BIC is administered to patients as a racemic mixture of the S and R isomers, we studied all three, racemic BIC as well as the S and R isomers. Co-crystallization of CYP46A1 with racemic BIC led to structure determinations at 2.1 Å resolution with the drug complexes exhibiting novel properties. Both BIC isomers bind to the CYP46A1 active site in very similar single orientation and adopt an energetically unfavorable folded conformation. This folded BIC conformation is correlated with drug-induced localized shifts of amino acid side chains in CYP46A1 and unusual interactions in the CYP46A1-BIC complex. One of these interactions involves a water molecule that is coordinated to the P450 heme iron and also hydrogen-bonded to the BIC nitrile. Due to polarization of the water in this environment, the heme elicits previously unreported types of P450 spectral responses. We also observed that access to the P450 active site was affected by differential recognition of S versus R isomers at the CYP46A1 surface arising from BIC conformational polymorphism.

Ligand access to the active site in Thermus thermophilus ba(3) and bovine heart aa(3) cytochrome oxidases.

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O(2) carriers, we recently found that NO and O(2) binding in Thermus thermophilus (Tt) ba(3) is ~10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallography, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild-type Tt ba(3) and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O(2) channel of Tt ba(3) are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa(3) enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba(3) and the T231F mutant. The results show that the Tt ba(3) Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic "way station", which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba(3) and bovine aa(3) show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba(3) Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-ethynylphenanthrene: implications for allosteric modulation of cytochrome P450 catalysis.

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k(inact) are 0.2 and 0.25 min(-1), respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S(50) of 4.5 µM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F' helix, ß1-ß2 loop, and ß4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.

The novel purification and biochemical characterization of a reversible CYP24A1:adrenodoxin complex.

Novel paradigms for CYP24A1 inhibitor development are needed to circumvent existing efficacy and toxicity issues related to human therapeutics in this class. We hypothesize that improved structural knowledge of CYP24A1 in complex with natural substrates, inhibitors and/or its redox partner protein, adrenodoxin (Adx) is required to facilitate the next generation of CYP24A1 inhibitor design. To this end, we have developed truncated expression constructs for both rat CYP24A1 (Δ51) and bovine Adx (Δ108), which allow us to purify a stable and reversible state of the CYP24A1:Adx complex, for use in ongoing X-ray crystallographic studies. Spectral characterization of the reversible complex revealed that Adx binding enhanced the stability of the enzyme-substrate complex, despite lowering the ligand binding affinity of the free enzyme, for 1,25(OH)2D2, over 9-fold. Truncation of CYP24A1's flexible N-terminus (Δ51) improved the enzyme's ability to recruit substrate, without altering Adx's ability to stabilize the ligand-bound form. We also found that several common crystallization detergents, including CHAPS, inhibit ligand binding to the CYP24A1:Adx complex at concentrations well below their reported critical micelle concentration (CMC) values. Ultimately, this research provides a useful platform and framework for the study of conformationally complex, membrane-protein complexes, in the ligand-bound state.