STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF MUTATIONS WITHIN THE HYDROPHOBIC CORES OF THE HMG1-BOX DOMAIN OF THE CHIRONOMUS HIGH-MOBILITY-GROUP PROTEIN 1A

The high-mobility-group protein 1 box domain (HMG1-BD) is a structural element found in several DNA-binding proteins in eukaryotic cells. It s structure is dominated by three a-helices. The spatial arrangement o f these helices into an L-shaped molecule is maintained by a number of apolar residues organized into a main and a secondary hydrophobic cor e. To analyze the significance of these residues for proper folding, c onformational stability, and ability to bind and bend DNA, we have mut ated the highly conserved Trp14 of the Chironomus HMG1a protein and ha ve synthesized a series of N-terminally truncated forms. The observed alterations in DNA-binding and DNA-bending characteristics were correl ated with structural consequences, as revealed by CD spectroscopy, lim ited trypsin digestion, and transverse urea gradient gel electrophores is. Mutation of the Trp14 residue (Chironomus [W14A]HMG1a) and deletio n of the seven N-terminal residues, respectively, which are members of the main and the secondary core of Chironomus HMG1a, both resulted in a substantial unfolding of the protein. Unexpectedly, these mutants s till retained their ability to bind and bend DNA. Conformational analy sis of wildtype cHMG1a and [W14A]cHMG1a showed that the proteins unfol d at 2-4 M urea. In contrast, their DNA complexes persisted even at 6- 8 M of the denaturant. Multiple contacts between the HMG1-BD and the D NA are probably responsible for the unusual stability of the complexes .