title: Probing site-specific interactions in protein-DNA complexes using heteronuclear NMR spectroscopy and molecular modeling: binding of Cro repressor to OR3.
abstract: 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 1H NMR and molecular modeling are used to develop a low resolution model and (b) 15N7-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 1H 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.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 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 operator DNA.
authors.affiliation: Structural Biology, Los Alamos National Laboratory, NM 87545, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Edwards
authors.forename: C S
authors.initials: CS
authors.lastname: Tung
authors.forename: L A
authors.initials: LA
authors.lastname: Silks
authors.forename: J M
authors.initials: JM
authors.lastname: Gatewood
authors.forename: J A
authors.initials: JA
authors.lastname: Fee
authors.forename: S V
authors.initials: SV
authors.lastname: Mariappan
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA