Probing site-specific interactions in protein-DNA complexes using heteronuclear NMR spectroscopy and molecular modeling: Binding of Cro repressor to OR sub(3)

In this paper, a general method is developed to study site-specific interactions in DNA-protein complexes using heteronuclear NMR spectroscopy and molecular modeling. This method involves two steps: (a) homonuclear super(1)H NMR and molecular modeling are used to develop a low resolution model and (...

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Bibliographic Details
Published in:Journal of biomolecular structure & dynamics 1998-08, Vol.16 (1), p.13-20
Main Authors: Edwards, CA, Tung, Chang-Shung, Silks, LA, Gatewood, J M, Fee, JA, Mariappan, SVS
Format: Article
Language:English
Subjects:
Phage lambda
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Summary:In this paper, a general method is developed to study site-specific interactions in DNA-protein complexes using heteronuclear NMR spectroscopy and molecular modeling. This method involves two steps: (a) homonuclear super(1)H NMR and molecular modeling are used to develop a low resolution model and (b) super(15)N7-guanosine containing oligonucleotides are employed to probe the specific intermolecular interactions predicted in (a). This method is applied to Cro-operator complex due to its small size and extensive prior characterization. Non-exchangeable and exchangeable base protons have been assigned by nuclear Overhauser effect spectroscopy (NOESY) and chemical shift correlation spectroscopy. Extensive line-broadening has been observed in the super(1)H NMR spectra of the operator DNA in the presence of protein. Differential line-broadening observed in the imino proton region and the comparison of NOESY spectra in the presence and absence of Cro protein show that guanosine-12 and guanosine-14 are involved in the Cro-DNA interaction, while the three A times T base-pairs at the 3'- and 5'-termini play only a minor role in the binding. A model of the Cro-operator DNA complex has been constructed by docking helix-3 of the Cro protein in the major groove and it predicted specific hydrogen bonds between N7 of guanosines-12 and -14 and the side-chain of Lys-32 and Set-28, respectively. The appearance of a new resonance in the temperature dependent proton detected heteronuclear multiple quantum coherence (HMQC) spectra of the Cro-DNA complex also demonstrates a specific interaction of Cro with guanosine-14 of the operator DNA.
ISSN:0739-1102