Charles E. Bell

Structural basis for antagonism and resistance of bicalutamide in prostate cancer

Carcinoma of the prostate is the most commonly diagnosed cancer in men. The current pharmacological treatment of choice for progressive androgen-dependent prostate cancer is the nonsteroidal antiandrogen, bicalutamide, either as monotherapy or with adjuvant castration or luteinizing hormone-releasing hormone superagonists to block the synthesis of endogenous testosterone. To date, no nonsteroidal or antagonist-bound androgen receptor (AR) structure is available. We solved the x-ray crystal structure of the mutant W741L AR ligand-binding domain bound to R-bicalutamide at 1.8-Å resolution. This mutation confers agonist activity to bicalutamide and is likely involved in bicalutamide withdrawal syndrome. The three-dimensional structure demonstrates that the B ring of R-bicalutamide in the W741L mutant is accommodated at the location of the indole ring of Trp-741 in the WT AR bound to dihydrotestosterone. Knowledge of the binding mechanism for R-bicalutamide will provide molecular rationale for the development of new antiandrogens and selective AR modulators.

A closer view of the conformation of the Lac repressor bound to operator.

Crystal structures of the Lac repressor, with and without isopropylthiogalactoside (IPTG), and the repressor bound to operator have provided a model for how the binding of the inducer reduces the affinity of the repressor for the operator. However, because of the low resolution of the operator-bound structure (4.8 Å), the model for the allosteric transition was presented in terms of structural elements rather than in terms of side chain interactions. Here we have constructed a dimeric Lac repressor and determined its structure at 2.6 Å resolution in complex with a symmetric operator and the anti-inducer orthonitrophenylfucoside (ONPF). The structure enables the induced (IPTG-bound) and repressed (operator-bound) conformations of the repressor to be compared in atomic detail. An extensive network of interactions between the DNA-binding and core domains of the repressor suggests a possible mechanism for the allosteric transition.

Crystal Structure of Diphtheria Toxin Bound to Nicotinamide Adenine Dinucleotide

The crystal structure of diphtheria toxin (DT) in complex with nicotinamide adenine dinucleotide (NAD) has been determined by x-ray crystallography to 2.3 A resolution. NAD binds to a cleft on the surface of the catalytic (C) domain of DT, interacting closely with the side chains of Tyr54, Tyr65, His21, Thr23, and Glu 48. The carboxylate group of Glu148 of Dt lies approximately 4 A from the scissile, N-glycosidic bound of NAD, suggesting a possible catalytic role for Glu148 in stabilizing a positively charged oxocarbonium intermediate. Residues 39-46 of the active-site loop of the C-domain become disordered upon NAD-binding, suggesting a potential role for these residues in binding to elongation facor-2 (EF-2). Structural alignments of the DT-NAD complex with the structures of other ADP-ribosylating toxins suggest how NAD may bind to these other enzymes.

Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

The mechanism by which the androgen receptor (AR) distinguishes between agonist and antagonist ligands is poorly understood. AR antagonists are currently used to treat prostate cancer. However, mutations commonly develop in patients that convert these compounds to agonists. Recently, our laboratory discovered selective androgen receptor modulators, which structurally resemble the nonsteroidal AR antagonists bicalutamide and hydroxyflutamide but act as agonists for the androgen receptor in a tissue-selective manner. To investigate why subtle structural changes to both the ligand and the receptor (i.e. mutations) result in drastic changes in activity, we studied structure-activity relationships for nonsteroidal AR ligands through crystallography and site-directed mutagenesis, comparing bound conformations of R-bicalutamide, hydroxyflutamide, and two previously reported nonsteroidal androgens, S-1 and R-3. These studies provide the first crystallographic evidence of the mechanism by which nonsteroidal ligands interact with the wild type AR. We have shown that changes induced to the positions of Trp-741, Thr-877, and Met-895 allow for ligand accommodation within the AR binding pocket and that a water-mediated hydrogen bond to the backbone oxygen of Leu-873 and the ketone of hydroxyflutamide is present when bound to the T877A AR variant. Additionally, we demonstrated that R-bicalutamide stimulates transcriptional activation in AR harboring the M895T point mutation. As a whole, these studies provide critical new insight for receptor-based drug design of nonsteroidal AR agonists and antagonists.

Crystal Structure of the λ Repressor C-Terminal Domain Provides a Model for Cooperative Operator Binding

Abstract Interactions between transcription factors bound to separate operator sites commonly play an important role in gene regulation by mediating cooperative binding to the DNA. However, few detailed structural models for understanding the molecular basis of such cooperativity are available. The c I repressor of bacteriophage λ is a classic example of a protein that binds to its operator sites cooperatively. The C-terminal domain of the repressor mediates dimerization as well as a dimer–dimer interaction that results in the cooperative binding of two repressor dimers to adjacent operator sites. Here, we present the x-ray crystal structure of the λ repressor C-terminal domain determined by multiwavelength anomalous diffraction. Remarkably, the interactions that mediate cooperativity are captured in the crystal, where two dimers associate about a 2-fold axis of symmetry. Based on the structure and previous genetic and biochemical data, we present a model for the cooperative binding of two λ repressor dimers at adjacent operator sites.

Crystal Structure of the T877A Human Androgen Receptor Ligand-binding Domain Complexed to Cyproterone Acetate Provides Insight for Ligand-induced Conformational Changes and Structure-based Drug Design

Cyproterone acetate (CPA) is a steroidal antiandrogen used clinically in the treatment of prostate cancer. Compared with steroidal agonists for the androgen receptor (AR) (e.g. dihydrotestosterone, R1881), CPA is bulkier in structure and therefore seemingly incompatible with the binding pockets observed in currently available x-ray crystal structures of the AR ligand-binding domain (LBD). We solved the x-ray crystal structure of the human AR LBD bound to CPA at 1.8Å in the T877A variant, a mutation known to increase the agonist activity of CPA and therefore facilitate purification and crystal formation of the receptor·drug complex. The structure demonstrates that bulk from the 17α-acetate group of CPA induces movement of the Leu-701 side chain, which results in partial unfolding of the C-terminal end of helix 11 and displacement of the loop between helices 11 and 12 in comparison to all other AR LBD crystal structures published to date. This structural alteration leads to an expansion of the AR binding cavity to include an additional pocket bordered by Leu-701, Leu-704, Ser-778, Met-780, Phe-876, and Leu-880. Further, we found that CPA invokes transcriptional activation in the L701A AR at low nanomolar concentrations similar to the T877A mutant. Analogous mutations in the glucocorticoid receptor (GR) and progesterone receptor were constructed, and we found that CPA was also converted into a potent agonist in the M560A GR. Altogether, these data offer information for structure-based drug design, elucidate flexible regions of the AR LBD, and provide insight as to how CPA antagonizes the AR and GR.

Structure and mechanism of Escherichia coli RecA ATPase.

RecA protein catalyses an ATP‐dependent DNA strand‐exchange reaction that is the central step in the repair of dsDNA breaks by homologous recombination. Although much is known about the structure of RecA protein itself, we do not at present have a detailed picture of how RecA binds to ssDNA and dsDNA substrates, and how these interactions are controlled by the binding and hydrolysis of the ATP cofactor. Recent studies from electron microscopy and X‐ray crystallography have revealed important ATP‐mediated conformational changes that occur within the protein, providing new insights into how RecA catalyses DNA strand‐exchange. A unifying theme is emerging for RecA and related ATPase enzymes in which the binding of ATP at a subunit interface results in large conformational changes that are coupled to interactions with the substrates in such a way as to promote the desired reactions.

The Lac repressor: a second generation of structural and functional studies

In the past year, the crystal structure of a dimeric version of the Escherichia coli Lac repressor bound to operator DNA was determined at 2.6A resolution, providing a closer view of the operator-bound conformation of the repressor. Refined NMR studies of the DNA-binding portion of the repressor complexed to operator DNA have revealed further details of the unique DNA-binding interactions of the repressor. The structural studies have been complemented by continued biochemical studies, with the overall goal of understanding the mechanism of allosteric regulation.

Substrate Orientation in Xanthine Oxidase CRYSTAL STRUCTURE OF ENZYME IN REACTION WITH 2-HYDROXY-6-METHYLPURINE

Xanthine oxidoreductase catalyzes the final two steps of purine catabolism and is involved in a variety of pathological states ranging from hyperuricemia to ischemia-reperfusion injury. The human enzyme is expressed primarily in its dehydrogenase form utilizing NAD+ as the final electron acceptor from the enzymes flavin site but can exist as an oxidase that utilizes O2 for this purpose. Central to an understanding of the enzymes function is knowledge of purine substrate orientation in the enzymes molybdenum-containing active site. We report here the crystal structure of xanthine oxidase, trapped at the stage of a critical intermediate in the course of reaction with the slow substrate 2-hydroxy-6-methylpurine at 2.3Å. This is the first crystal structure of a reaction intermediate with a purine substrate that is hydroxylated at its C8 position as is xanthine and confirms the structure predicted to occur in the course of the presently favored reaction mechanism. The structure also corroborates recent work suggesting that 2-hydroxy-6-methylpurine orients in the active site with its C2 carbonyl group interacting with Arg-880 and extends our hypothesis that xanthine binds opposite this orientation, with its C6 carbonyl positioned to interact with Arg-880 in stabilizing the MoV transition state.

Crystal structures of λ exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity

The λ exonuclease is an ATP-independent enzyme that binds to dsDNA ends and processively digests the 5′-ended strand to form 5′ mononucleotides and a long 3′ overhang. The crystal structure of λ exonuclease revealed a toroidal homotrimer with a central funnel-shaped channel for tracking along the DNA, and a mechanism for processivity based on topological linkage of the trimer to the DNA was proposed. Here, we have determined the crystal structure of λ exonuclease in complex with DNA at 1.88-Å resolution. The structure reveals that the enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5′-ended strand insert into the active site of one subunit of the trimer, while the 3′-ended strand passes through the central channel to emerge out the back of the trimer. Unwinding of the DNA is facilitated by several apolar residues, including Leu78, that wedge into the base pairs at the single/double-strand junction to form favorable hydrophobic interactions. The terminal 5′ phosphate of the DNA binds to a positively charged pocket buried at the end of the active site, while the scissile phosphate bridges two active site Mg2+ ions. Our data suggest a mechanism for processivity in which wedging of Leu78 and other apolar residues into the base pairs of the DNA restricts backward movement, whereas attraction of the 5′ phosphate to the positively charged pocket drives forward movement of the enzyme along the DNA at each cycle of the reaction. Thus, processivity of λ exonuclease operates not only at the level of the trimer, but also at the level of the monomer.