PROBING SITE-SPECIFIC INTERACTIONS IN PROTEIN-DNA COMPLEXES USING HETERONUCLEAR NMR-SPECTROSCOPY AND MOLECULAR MODELING - BINDING OF CRO REPRESSOR TO OR3

In this paper, a general method is developed to study site-specific in teractions in DNA-protein complexes using heteronuclear NMR spectrosco py and molecular modeling. This method involves two steps: (a) homonuc lear H-1 NMR and molecular modeling are used to develop a low resoluti on model and (b) (15)N7-guanosine containing oligonucleotides are empl oyed 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 exchangeab le base protons have been assigned by nuclear Overhauser effect spectr oscopy (NOESY) and chemical shift correlation spectroscopy. Extensive line-broadening has been observed in the H-1 NMR spectra of the operat or DNA in the presence of protein. Differential line-broadening observ ed in the imino proton region and the comparison of NOESY spectra in t he presence and absence of Cro protein show that guanosine-12 and guan osine-14 are involved in the Cro-DNA interaction, while the three A.T base-pairs at the 3'- and 5'-termini play only a minor role in the bin ding. 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 predicte d specific hydrogen bonds between N7 of guanosines-12 and -14 and the side-chain of Lys-32 and Ser-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 op erator DNA.