Proton NMR study of the [d(ACGTATACGT)]2-2echinomycin complex: conformational changes between echinomycin binding sites

Nucleic Acids Research, Vol. 20, No. 10 2411-2420 Proton NMR study of the [d(ACGTATACGT)]2-2echinomycin complex: conformational changes between echinomycin binding sites Dara E.Gilbert+ and Juli Feigon* Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90024, USA Received April 18, 1992; Accepted April 21, 1992 The interactions of echinomycin and the DNA decamer [d(ACGTATACGT)]2 were studied by proton NMR. Echinomycin binds cooperatively as a bisintercalator at the CpG steps. The terminal A-T base pairs are Hoogsteen base paired, but none of the four central A-T base pairs are Hoogsteen base paired. However, binding of the drug induces unwinding of the DNA which is propagated to the central ApT step. All four central A • T base pairs are destabilized relative to those in the free DNA. Furthermore, based on these and other results from our laboratory, we conclude that the formation of stable Hoogsteen base pairs may not be the relevant structural change in vivo. The structural changes propagated between adjacent ACGT binding sites are the unwinding of the duplex and destabilization of the base pairing between binding sites. INTRODUCTION Echinomycin is a naturally occurring antibiotic comprised of an octadepsipeptide ring with a thioacetal cross bridge between opposing cysteines and quinoxaline rings attached via peptide linkages to the two D-serine residues (Chart I). It exhibits potent anti-viral and anti-tumor activity (1). Echinomycin is currently in phase two clinical trials as a chemotherapeutic agent (2). Footprinting experiments established the preference for binding of echinomycin at CpG steps (3,4) and showed that echinomycin binding altered the structure of the DNA adjacent to the echinomycin binding sites, rendering it more reactive with DNase I and other DNA cleavage reagents (3, 5). This hyperreactivity to cleavage reagents is also observed several base pairs away from the nearest binding site. Mendel and Dervan (5) and Fox and Kentebe (6) showed that binding of echinomycin to DNA causes hyperreactivity to DNase I or DEPC up to 12 base pairs from the nearest binding site. The structural basis for the enhanced reactivity of DNA distal to the binding sites is not well understood. McClean and co-workers showed that DNA fragments in which the adenines were modified at the N7 position such that they could not Hoogsteen base pair remained hypersensitive to cleavage by OsO4 (7). Based on these results, they proposed that this structural change was the unwinding of the DNA duplex induced by the intercalative binding of echinomycin rather than the formation of Hoogsteen base pairs (7). None of the structural studies to date have addressed the issue of what structural changes are propagated between binding sites several base pairs apart. No structural information, either in solution or the crystalline state, has been obtained for DNAechinomycin complexes in which two CpG binding sites are separated by more than two A • T base pairs. In order to address questions regarding the structure of the DNA between two echinomycin binding sites, the complex formed between echinomycin and [d(ACGTATACGT)]2, in which the CpG binding sites are separated by four base pairs, was studied by 'H NMR. The goals of this study were to determine: (1) if Hoogsteen base pairs form; (2) if so, are Hoogsteen base pairs propagated one base pair away from the binding site; and (3) if not, what other structural changes occur that account for the observed hyperreactivity to DNA cleavage reagents of DNA distal to the binding site. Many of the results obtained in this study are consistent with those obtained on the [d(ACGTACGT)]2-2echinomycin * To whom correspondence should be addressed + Present address: Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, NL-3584 CH, Utrecht, The Netherlands ABSTRACT 2412 Nucleic Acids Research, Vol. 20, No. 10 complex. Two drugs bind cooperatively per duplex, one at each of the CpG steps. The terminal A T base pairs are Hoogsteen base paired. All four central A T base pairs are destabilized relative to the free DNA. However, in contrast to the results obtained for the [d(ACGTACGT)]2-2echinomycin complex, there is no direct evidence for the formation of Hoogsteen base pairs between the binding sites at any temperature. There is evidence that the helix is unwound through the four central A • T base pairs. Two structural changes, destabilization of base pairing and unwinding of the duplex, are propagated through all four A-T base pairs between the two echinomycin intercalation sites. NMR Spectroscopy All NMR experiments were done on a General Electric GN500 (500.119 MHz, 'H) spectrometer (GE NMR, Fremont, CA). Chemical shifts were referenced to the chemical shift of water, which had been previously calibrated relative to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). Phase sensitive nuclear Overhauser effect (NOESY) spectra in D2O were obtained using the method of States et al. (15) and the standard RESULTS Complex Formation Figure 1 shows the aromatic region of the spectrum of [d(ACGTATACGT)]2-echinomycin as a function of increasing drug:DNA ratio. Upon addition of 0.5 equivalents of drug, a second set of resonances appears. As the drug:DNA ratio is raised, the intensity of the second set of lines increases and the resonances from the free DNA decrease in intensity until, at a drug:DNA ratio of 2:1, only the resonances from the fully saturated complex are observed. The appearance of two sets of resonances indicates that the free and drug complexed DNA are >DRUG 1-2 I 5 Figure 1. Proton NMR spectra of the aromatic region of [d(ACGTATACGT)]2-echinomycin as a function of added drug concentration at 30°C in D2O. DNA:echinomycin ratios of (A) 1:0, (B) 1:0.5, (C)l:l, and (D) 1:2 are shown. Solid lines connect the free DNA resonances to the free DNA resonances in the spectra of the 1:0 and 1:0.5 complexes; dashed lines connect the resonances of the fully saturated complex with the resonances of the fully saturated complex in the spectrum of the 1:1 complex. 0.5 equivalents of echinomycin in methanol were added to the DNA (1.9 mM duplex, 104 mM NaCl, pH 6.6) in the NMR tube. After each addition, the sample was dried under a stream of N2 gas and redissolved in 99.996% D2O. The spectra were acquired with a sweep width of 5000 Hz in 8K complex points. Data were Gaussian broadened by 3Hz prior to Fourier transformation. MATERIALS AND METHODS Sample Preparation The DNA decamer d(A|C2G3T 4 A 5 T 6 A 7 C 8 G 9 T 10 ) was synthesized on the 10 /oriole scale on an Applied Biosystems 381A DNA synthesizer using phosphoramidite chemistry. The decamer was purified by gel filtration as previously described (13). Two DNA samples were prepared by dissolving the DNA in 50 mM NaCl and adjusting the pH to 6.5 (uncorrected meter reading) with 0.1 M NaOH. No additional (...truncated)