X-Ray Crystallographic Studies of DNA-Drug and DNA-Protein Interactions

Citation

Pjura, Philip (1987) X-Ray Crystallographic Studies of DNA-Drug and DNA-Protein Interactions. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/wpt7-9h98. https://resolver.caltech.edu/CaltechTHESIS:10232019-113724052

Abstract

I. Cisplatin-12mer

Cisplatin (cis-diamminodichloroplatinum[II]) is a widely used antineoplastic agent, which is believed to work by means of covalent interaction with DNA. Complexes of this compound were made with the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G by diffusion of the drug into pregrown DNA crystals, and a structure determined to 2.6 Angstrom resolution by molecular replacement. Cisplatin was found to bind with partial occupancy at three discrete sites: G16 (61%), G4 (30%) and G10 (22%), in each case by means of a single covalent bond from the metal to guanine N7 in the major groove. The square plane of the metal complex ligands is rotated out of the plane of the guanine base, with one of the ligands that is cis to the guanine N7, presumably an amine, in a position to make a hydrogen bond with guanine 06; the long metal-06 distance precludes the possiblilty of a direct metal-06 bond. The DNA structure itself is essentially undisturbed by the metal binding; the only change is a slight notion of the bound guanines outward into the major groove toward the metals, resulting in a slight opening up of the groove but without pulling the base-pairs out of the helix stack. The structure shows that it is not possible for a direct N7-to-N7 crosslink between two adjacent bases by the metal to exist in an intact B-DNA double-helix. It is suggested that the observed structure is a primary mode of binding of the drug, which then could become this postulated active form upon disruption of the DNA duplex.

II. Lac headpiece-operator complex

The lac repressor protein of E. coli controls expression of the genes necessary for lactose utilization by the organism. It is a tetramer of four identical subunits of 355 amino acids each, each of which is divided into a 51 amino acid N-terminal DNA-binding, or "headpiece" domain and a 300 residue C-terminal regulatory region, or "core." In the presence of 1M Tris.HCl pH 7.5 and 30% glycerol, the headpiece can be isolated intact by proteolytic digestion and is believed to retain its specific DNA binding properties for the lac operator site. A large-scale purification scheme for the headpiece protein has been developed, using a specific protease-affinity column to eliminate all residual proteolytic activity from the prep, thus making it possible to isolate large quantities of the protease-sensitive fragment in stable form for crystallization trials. Attempts to crystallize the protein by itself resulted only in fibrous microcrystals. But cocrystallization trials with a 21 base-pair lac operator DNA oligomer yielded what appear to be small cocrystals, too small to characterize, under conditions in which neither the protein nor the DNA by themselves would crystallize.

III. Hoechst 33258-Complex with DNA 12mer

Hoechst 33258 is a widely used histological stain that forms a fluorescent complex with DNA, showing strong preference for AT-rich regions. Crystals were grown of a 1:1 complex of the dye with the B-DNA 12rner C-G-C-G-A-A-T-T-C-G-C-G, and a structure was determined to 2.2 Angstrom resolution. The compound was found to bind noncovalently in the minor groove, with its aromatic phenol and two benzimidazole rings spanning three bases of the A-A-T-T region, and its aliphatic piperazine ring binding in the adjacent C-G-C-G region. Three hydrogen bond contacts were found to bridge between adjacent base-pairs, the bridging resembling that by water molecules in the native structure. This bridging of base pairs was achieved via three amine groups on the dye. These all undoubtedly help to stabilize the interaction, but the shape of the compound allows a good hydrogen bonding interaction of normal length through only one of these contacts. The piperazine ring, because of its orientation perpendicular to the other rings, is unable to bind into the narrower minor groove of the AT region and must bind in the wider CG minor groove. In a second conformation of the dye, also seen in the structure, the piperazine ring points out of the minor groove because of a rotation of the benzimidazole ring to which it is attached around its bond to the other benzimidazole. In this orientation, the piperazine ring no longer makes contact with the DNA, and therefore the drug is not restricted to binding at a site containing GC base-pairs. As a result, it could bind within a region of contiguous AT base-pairs without the problem of steric clash due to the piperazine ring.

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