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.

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.

Summary: 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 lambda 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. 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 lambda exonuclease in complex with DNA at 1.88-angstrom 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 lambda exonuclease operates not only at the level of the trimer, but also at the level of the monomer.