Structural and functional consequences of mutations within the hydrophobic cores of the HMG1-box domain of the Chironomus high-mobility-group protein 1a.

Abstract

The high-mobility-group protein 1 box domain (HMG1-BD) is a structural element found in several DNA-binding proteins in eukaryotic cells. Its structure is dominated by three alpha-helices. The spatial arrangement of these helices into an L-shaped molecule is maintained by a number of apolar residues organized into a main and a secondary hydrophobic core. To analyze the significance of these residues for proper folding, conformational stability, and ability to bind and bend DNA, we have mutated the highly conserved Trp14 of the Chironomus HMG1a protein and have synthesized a series of N-terminally truncated forms. The observed alterations in DNA-binding and DNA-bending characteristics were correlated with structural consequences, as revealed by CD spectroscopy, limited trypsin digestion, and transverse urea gradient gel electrophoresis. Mutation of the Trp14 residue (Chironomus [W14A]HMG1a) and deletion 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 still retained their ability to bind and bend DNA. Conformational analysis of wild-type cHMG1a and [W14A]cHMG1a showed that the proteins unfold 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 DNA are probably responsible for the unusual stability of the complexes.