1H NMR study of [d(GCGATCGC)]2 and its interaction with minor groove binding 4',6-diamidino-2-phenylindole

Two-dimensional NMR spectroscopy has been applied to study the solution binding of 4',6-diamidino-2-phenylindole (DAPI) to synthetic DNA duplex [d(GCGATCGC)]2. The structure of the complex at a molar ratio of 1:1 drug:duplex has been investigated. NMR results indicate that DAPI binds selectively in the minor groove of the DNA region containing only two A:T base pairs. The results disagree with conclusions drawn from footprinting experiments and show that the presence of the G3NH2 group in the minor groove does not prevent the binding. A molecular model is proposed that closely resembles the crystal structure previously published for the interaction of DAPI with the dodecamer [d(CGCGAATTCGCG)]2, containing four A:T base pairs in the binding site. In this model, DAPI lies in the minor groove, nearly isohelical, with its aromatic rings adjacent to H4' protons of T5 and C6 deoxyribose and the NH indole group oriented toward the DNA axis. The binding does not perturb the B-type conformation of the duplex, and the DNA oligomer conserves its 2-fold symmetry, indicating that fast exchange dynamics exist between the two stereochemically equivalent binding sites of the palindromic sequence. The binding constant and the exchange rate between free and bound species were also measured by NMR spectroscopy.     

A helix-turn-helix structure unit in human centromere protein B (CENP-B)

CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically binds to the CENP-B box sequence (17 bp) in centromere DNA. We determined the solution structure of human CENP-B DBD RP1 by multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found. The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP-B DBD RP1. We studied the interaction between CENP-B DBD RP1 and the CENP-B box by the use of NMR chemical shift perturbation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B box DNA, mainly at the N-terminal basic region, the N-terminal portion of helix 2 and helix 3.     

Solution structure of human intestinal fatty acid binding protein: implications for ligand entry and exit

The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy. Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra (NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel beta-strands which form two nearly orthogonal beta-sheets of five strands each, and two short alpha-helices that connect the beta-strands A and B. The interior of the protein consists of a water-filled cavity between the two beta-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP. The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand.     

Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR

The paramagnetic metal ion Mn2+ has been used to probe the electrostatic potentials of a DNA quadruplex that has two quartets with an overall fold of the chair type. A quadruplex with a basket type structure has also been examined. The binding of the paramagnetic ion manganese to these quadruplex DNAs has been investigated by solution state electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies. The EPR results indicate that the DNA aptamer, d(GGTTGGTGTGGTTGG), binds two manganese ions and that the binding constants for each of these sites is approximately 10(5) M-1. The NMR results indicate that the binding sites of the manganese are in the narrow grooves of this quadruplex DNA. The binding sites of the DNA quadruplex formed by dimers of d(GGGGTTTTGGGG) which forms a basket structure are also in the narrow groove. These results indicate that the close approach of phosphates in the narrow minor grooves of the quadruplex structures provide strong binding sites for the manganese ions and that EPR and NMR monitoring of manganese binding can be used to distinguish between the different types of quadruplex structures.     

NMR structure of the extended Myb cognate sequence and modeling studies on specific DNA-Myb complexes

The recognition sequence of the Myb protein has been recently described to be pyAACKGHH (where py = T/C, K = G/T, and H = A/C/T), modifying the earlier identification as pyAACKG [Ording, E., et al. (1994) Eur. J. Biochem. 222, 113-120]. We had earlier determined the solution structure of the minimal cognate sequence TAACGG, choosing py = T and K = G, embeded in a 12-mer DNA duplex by NMR and related computational techniques [Radha, P. K., et al. (1995) Biochemistry 34, 5913-5912]. To understand the structural significance of the above modification and the role of the variability in the recognition sequence, we have investigated here the solution structure of a different DNA segment, d-ACAACTGCAGTTGT, which contains the extended Myb cognate site, CAACTGCA. The three-dimensional structure of the 14-mer duplex has been determined from NMR data by relaxation matrix and restrained molecular dynamics calculations. The structure of the above cognate sequence in the 14-mer duplex has been compared with that of the cognate sequence, TAACGG, in the 12-mer duplex and also with that in the NMR structure of the Myb DNA binding domain (R2R3)-DNA complex determined by Ogata et al. recently [Ogata, K., et al. (1994) Cell 79, 639-648]. The comparison highlighted differences in several structural parameters for the cognate sites in the DNA segments. Modeling studies by taking out the protein from the complex and presenting it with 12-mer and 14-mer DNA structures indicated that the protein induces structural alterations to drive the cognate site to a reasonably conserved structure. The extent of similarity of the derived structures was, however, dependent on the base sequences. Base changes in the minimal cognate sequence in the 12-mer-protein complex and in the 14-mer-protein complex so as to match the sequence of Ogata et al. produced a more conserved structure of the complex. A reverse exercise, in which the Ogata DNA in the complex was mutated to match the 12-mer and 14-mer minimal cognate sequences, complemented the above observations of the subtle sequence dependence of the structure in the complex. On the other hand, base changes in the extension did not influence the DNA-protein complex structure significantly. We also observed that the structural changes in the protein were very minor when different DNA sequences or different DNA structures were presented to it. These observations would be of interest from the point of view of DNA-Myb recognition.