Two distinct rotations of bithiazole DNA intercalation revealed by direct comparison of crystal structures of Co(III)•bleomycin A2 and B2 bound to duplex 5'-TAGTT sites.

Bleomycins constitute a family of anticancer natural products that bind DNA through intercalation of a C-terminal tail/bithiazole moiety and hydrogen-bonding interactions between the remainder of the drug and the minor groove. The clinical utility of the bleomycins is believed to result from single- and double-strand DNA cleavage mediated by the HOO-Fe(III) form of the drug. The bleomycins also serve as a model system to understand the nature of complex drug-DNA interactions that may guide future DNA-targeted drug discovery. In this study, the impact of the C-terminal tail on bleomycin-DNA interactions was investigated. Toward this goal, we determined two crystal structures of HOO-Co(III)•BLMA2 "green" (a stable structural analogue of the active HOO-Fe(III) drug) bound to duplex DNA containing 5'-TAGTT, one in which the entire drug is bound (fully bound) and a second with only the C-terminal tail/bithiazole bound (partially bound). The structures reported here were captured by soaking HOO-Co(III)•BLMA2 into preformed host-guest crystals including a preferred DNA-binding site. While the overall structure of DNA-bound BLMA2 was found to be similar to those reported earlier at the same DNA site for BLMB2, the intercalated bithiazole of BLMB2 is "flipped" 180˚ relative to DNA-bound BLMA2. This finding highlights an unidentified role for the C-terminal tail in directing the intercalation of the bithiazole. In addition, these analyses identified specific bond rotations within the C-terminal domain of the drug that may be relevant for its reorganization and ability to carry out a double-strand DNA cleavage event.

Crystal structure of the human Hsmar1-derived transposase domain in the DNA repair enzyme Metnase.

Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.

Crystal structure of a trypanocidal 4,4'-bis(imidazolinylamino)diphenylamine bound to DNA.

The pursuit of small molecules that bind to DNA has led to the discovery of selective and potent antitrypanosomal agents, specifically 4,4'-bis(imidazolinylamino)- and 4,4'-bis(guanidino)diphenylamine compounds, CD27 and CD25, respectively. Although the antitrypanosomal properties of these compounds have been characterized, further development of this series of compounds requires assessment of their DNA site selectivities and affinities. Toward this end, both compounds have been analyzed and found to selectively bind AT sequences. However, CD27 was found to bind with higher affinity to 5'-AATT than 5'-ATAT while CD25 bound more weakly but equally well to either sequence. To detail the nature of its interactions with DNA, the crystal structure of CD27, bound to its preferred DNA-binding site 5'-AATT within a self-complementary oligonucleotide, 5'-d(CTTAATTCGAATTAAG), was determined at 1.75 A using a host-guest approach. Although CD27 is predicted to be highly twisted in its energy-minimized state, it adopts a more planar crescent shape when bound in the minor groove of the DNA. Interactions of CD27 with 5'-AATT include bifurcated hydrogen bonds, providing a basis for selectivity of this site, and favorable van der Waals interactions in a slightly widened minor groove. Thus, an induced fit results from conformational changes in both the ligand and the DNA. Our studies suggest a basis for understanding the mechanism of the antitrypanosomal activity of these symmetric diphenylamine compounds.

Crystal structure of DNA-bound Co(III) bleomycin B2: Insights on intercalation and minor groove binding.

Bleomycins constitute a widely studied class of complex DNA cleaving natural products that are used to treat various cancers. Since their first isolation, the bleomycins have provided a paradigm for the development and discovery of additional DNA-cleaving chemotherapeutic agents. The bleomycins consist of a disaccharide-modified metal-binding domain connected to a bithiazole/C-terminal tail via a methylvalerate-Thr linker and induce DNA damage after oxygen activation through site-selective cleavage of duplex DNA at 5'-GT/C sites. Here, we present crystal structures of two different 5'-GT containing oligonucleotides in both the presence and absence of bound Co(III).bleomycin B(2). Several findings from our studies impact the current view of bleomycin binding to DNA. First, we report that the bithiazole intercalates in two distinct modes and can do so independently of well ordered minor groove binding of the metal binding/disaccharide domains. Second, the Co(III)-coordinating equatorial ligands in our structure include the imidazole, histidine amide, pyrimidine N1, and the secondary amine of the beta aminoalanine, whereas the primary amine acts as an axial ligand. Third, minor groove binding of Co(III).bleomycin involves direct hydrogen bonding interactions of the metal binding domain and disaccharide with the DNA. Finally, modeling of a hydroperoxide ligand coordinated to Co(III) suggests that it is ideally positioned for initiation of C4'-H abstraction.

Structural basis for regulation of protein phosphatase 1 by inhibitor-2.

The functional specificity of type 1 protein phosphatases (PP1) depends on the associated regulatory/targeting and inhibitory subunits. To gain insights into the mechanism of PP1 regulation by inhibitor-2, an ancient and intrinsically disordered regulator, we solved the crystal structure of the complex to 2.5A resolution. Our studies show that, when complexed with PP1c, I-2 acquires three regions of order: site 1, residues 12-17, binds adjacent to a region recognized by many PP1 regulators; site 2, amino acids 44-56, interacts along the RVXF binding groove through an unsuspected sequence, KSQKW; and site 3, residues 130-169, forms alpha-helical regions that lie across the substrate-binding cleft. Specifically, residues 148-151 interact at the catalytic center, displacing essential metal ions, accounting for both rapid inhibition and slower inactivation of PP1c. Thus, our structure provides novel insights into the mechanism of PP1 inhibition and subsequent reactivation, has broad implications for the physiological regulation of PP1, and highlights common inhibitory interactions among phosphoprotein phosphatase family members.

Unusually strong binding to the DNA minor groove by a highly twisted benzimidazole diphenylether: induced fit and bound water.

RT29 is a dicationic diamidine derivative that does not obey the classical "rules" for shape and functional group placement that are expected to result in strong binding and specific recognition of the DNA minor groove. The compound contains a benzimidazole diphenyl ether core that is flanked by the amidine cations. The diphenyl ether is highly twisted and gives the entire compound too much curvature to fit well to the shape of the minor groove. DNase I footprinting, fluorescence intercalator displacement studies, and circular dichroism spectra, however, indicate that the compound is an AT specific minor groove binding agent. Even more surprisingly, quantitative biosensor-surface plasmon resonance and isothermal titration calorimetric results indicate that the compound binds with exceptional strength to certain AT sequences in DNA with a large negative enthalpy of binding. Crystallographic results for the DNA complex of RT29 compared to calculated results for the free compound show that the compound undergoes significant conformational changes to enhance its minor groove interactions. In addition, a water molecule is incorporated directly into the complex to complete the compound-DNA interface, and it forms an essential link between the compound and base pair edges at the floor of the minor groove. The calculated DeltaCp value for complex formation is substantially less than the experimentally observed value, which supports the idea of water being an intrinsic part of the complex with a major contribution to the DeltaCp value. Both the induced fit conformational changes of the compound and the bound water are essential for strong binding to DNA by RT29.

A high-throughput, high-resolution strategy for the study of site-selective DNA binding agents: analysis of a "highly twisted" benzimidazole-diamidine.

A general strategy for the rapid structural analysis of DNA binding ligands is described as it was applied to the study of RT29, a benzimidazole-diamidine compound containing a highly twisted diphenyl ether linkage. By combining the existing high-throughput fluorescent intercalator displacement (HT-FID) assay developed by Boger et al. and a high-resolution (HR) host-guest crystallographic technique, a system was produced that was capable of determining detailed structural information pertaining to RT29-DNA interactions within approximately 3 days. Our application of the HT/HR strategy immediately revealed that RT29 has a preference for 4-base pair (bp), A.T-rich sites (AATT) and a similar tolerance and affinity for three A-T-bp sites (such as ATTC) containing a G.C bp. On the basis of these selectivities, oligonucleotides were designed and the host-guest crystallographic method was used to generate diffraction quality crystals. Analysis of the resulting crystal structures revealed that the diphenyl ether moiety of RT29 undergoes conformational changes that allow it to adopt a crescent shape that now complements the minor groove structure. The presence of a G.C bp in the RT29 binding site of ATTC did not overly perturb its interaction with DNA-the compound adjusted to the nucleobases that were available through water-mediated interactions. Our analyses suggest that the HT/HR strategy may be used to expedite the screening of novel minor groove binding compounds leading to a direct, HR structural determination.

A host-guest approach for determining drug-DNA interactions: an example using netropsin.

Netropsin is a well-characterized DNA minor groove binding compound that serves as a model for the study of drug-DNA interactions. Our laboratory has developed a novel host-guest approach to study drug-DNA interactions in which the host, the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase (MMLV RT) is co-crystallized with a DNA oligonucleotide guest in the presence and absence of drug. We have co-crystallized netropsin with the RT fragment bound to the symmetric 16mer d(CTTAATTCGAATTAAG)2 and determined the structure of the complex at 1.85 A. In contrast to previously reported netropsin-DNA structures, our oligonucleotide contains two AATT sites that bind netropsin with flanking 5' and 3' sequences that are not symmetric. The asymmetric unit of the RT fragment-DNA-netropsin crystals contains one protein molecule and one-half of the 16mer with one netropsin molecule bound. The guanidinium moiety of netropsin binds in a narrow part of the minor groove, while the amidinium is bound in the widest region within the site. We compare this structure to other Class I netropsin-DNA structures and find that the asymmetry of minor groove widths in the AATT site contributes to the orientation of netropsin within the groove while hydrogen bonding patterns vary in the different structures.