Molecular characterization of a tetramolecular complex between dsDNA and aDNA-binding leucine zipper peptide dimer by mass spectrometry

The characterization of sequence-specific noncovalent complexes of the GCN4 peptides and dsDNA using mass spectrometry is reported. The GCN4 peptides belong to a class of proteins which bind to sequence-specific dsDNA and are important in the regulation of gene transcription in yeast. These proteins contain a bZIP structural motif which consists of a basic DNA-binding doma in and a leucine zipper dimerization domain. The protein dimers specificall y bind double-stranded DNA containing the binding element 5'-ATGA(C/G)TCAT- 3' to form a tetramolecular noncovalent complex. Using electrospray ionizat ion, we report the detection of such a specific tetramolecular complex usin g mass spectrometry. Under conditions necessary for observation of the tetr amolecular complex, no ions were detected far the GCN4 peptide dimer or the GCN4 monomer with dsDNA. These observations indicate that the specific int eraction of the dsDNA with the protein dimer stabilizes the biologically si gnificant noncovalent complex in the gas phase. Complexes were observed for various lengths of both blunt-ended and cohesive-ended double-stranded DNA containing the specific recognition sequence. The binding specificity of t he complex was verified with the use of control DNA not containing the reco gnition sequence and control peptides not known to bind DNA specifically. A dditionally, combining limited proteolysis of GCN4 peptide-DNA complexes wi th mass spectrometric determination of the products compared to identical e xperiments with noncomplexed peptides was used to probe interactions of spe cific amino acids with the DNA. The ability to observe these complexes by m ass spectrometry. and to probe the specific interactions involved opens the door for utilizing this analytical technique to other structural biologica l problems including the study of transcription processes and determining t he specific binding regions between dsDNA and proteins.