Structural insights into the catalytic mechanism of human topoisomerase I and the crystal structure of the human PXR ligand binding domain with the macrolide rifampicin

Human topoisomerase I relaxes superhelical tension associated with DNA replication, transcription and recombination by reversibly nicking one strand of duplex DNA and forming a covalent 3'-phosphotyrosine linkage. The enzyme is the sole target of the camptothecin family of anticancer drugs which act by reversibly stabilizing the covalent topoisomerase I-DNA complex, resulting in double-strand DNA breaks and apoptosis. Three crystal structures of human topoisomerase I in complexes with anticancer drugs are presented. The first is of human topoisomerase I in trapped covalent complex with a DNA duplex containing an 1-beta-D-arabinofuranosylcytosine (Ara-C) base. The presence of Ara-C in DNA was found to distort the DNA double-helix, and these distortions are translated from the DNA to the active site of human topoisomerase I. The free sulfhydryl at the 5'-end of the nicked DNA strand in this trapped covalent complex is shifted out of alignment with the 3'-phosphotyrosine linkage at the catalytic tyrosine-723 residue, producing a geometry not optimal for religation. The second two structures are ternary complexes of camptothecin-resistant mutant forms of human topoisomerase I (Asn-722-Ser and Phe-361-Ser) in with duplex DNA and the camptothecin analogue topotecan.; These structures reveal that while the Asparagine-722 to serine mutation eliminates a water-mediated contact between the topoisomerase I and topotecan, the Phenyalanine-361 to serine mutation requires a water molecule to be buried to replace the hydrophobic phenylalanine side chain and to allow for topotecan binding.; The human pregnane X receptor (PXR) has been identified as the master regulator of the expression of the CYP3A4 gene, which encodes for the enzyme responsible for metabolizing more than 50% of all prescription drugs. The crystal structure of the human PXR ligand binding domain in complex with the macrolide rifampicin is presented. The structure reveals that the PXR ligand binding pocket swells to accommodate rifampicin to more than 1600 A3 in size, making it the largest binding cavity described for a nuclear receptor to date. This structure provides insight into the promiscuous ability of PXR to bind to structurally-distinct ligands, and the ability of PXR's from different species to be selective as well as promiscuous in terms of ligand binding.