Identification of the regions involved in DNA binding by the mouse PEBP2 alpha protein

The polyomavirus enhancer binding protein 2 alpha (PEBP2 alpha) is a DNA bi nding transcriptional regulatory protein that binds conserved sites in the polyomavirus enhancer, mammalian type C retroviral enhancers and T-cell rec eptor gene enhancers. Binding of PEBP2 alpha and homologous proteins to the consensus DNA sequence TGPyGGTPy is mediated through a protein domain know n as the runt domain. Although recent NMR studies of DNA-bound forms of the runt domain have shown an immunoglobulin-like (Ig) fold, the identificatio n of residues of the protein that are involved in DNA binding has been obsc ured by the low solubility of the runt domain, Constructs of the mouse PEBP 2 alpha A1 gene were generated with N- and C-terminal extensions beyond the runt homology region, The construct containing residues Asp90 to Lys225 of the sequence (PEBP2 alpha 90-225) yielded soluble protein. The residues th at participate in DNA binding were determined by comparing the NMR spectra of free and DNA-bound PEBP2 alpha 90-225, Analysis of the changes in the NM R spectra of the two forms of the protein by chemical shift deviation mappi ng allowed the unambiguous determination of the regions that are responsibl e for specific DNA recognition by PEBP2 alpha. Five regions in PEBP2 alpha 90-225 that are localized at one end of the beta-barrel were found to inter act with DNA, similar to the DNA binding interactions of other Ig fold prot eins. (C) 2000 Federation of European Biochemical Societies.