Replacement of conserved threonines by alanine residues in high mobility group protein HMG-I(Y): effect on DNA binding affinity.
A threonine residue at the beginning of each DNA-binding domain of HMG-I (residue numbers 21, 53, and 78) is conserved among mammalian species and proposed to help stabilize the A.T-hook DNA-binding motif. Phosphorylation of threonines number 53 and 78 of human HMG-I(Y) both in vivo and in vitro leads to a 20 fold reduction in the proteins DNA binding affinity. Recombinant human HMG-I proteins were engineered to contain alanine instead of the conserved threonine in each DNA-binding domain. The DNA dissociation constant of each protein was assayed at various salt concentrations by competition with the fluorescent dye Hoechst 33258 for an AT-rich DNA substrate. Replacement of these threonines did not affect the equilibrium binding of these proteins to DNA as compared with wild-type HMG-I and HMG-Y. Molecular modelling of analogous peptides supported this finding. We conclude that these threonines are not directly important for A.T-hook DNA-binding and are conserved phosphorylation sites for down regulation of DNA binding by the A.T-hook motif in the HMG-I(Y) proteins.
Siino
J S
JS
Department of Biophysics and Biochemistry, Washington State University, Pullman 99164-4660.
Nissen
M S
MS
Reeves
R
R
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D006690:Bisbenzimidazole; D003001:Cloning, Molecular; D003198:Computer Simulation; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D025741:HMGA1a Protein; D006609:High Mobility Group Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013912:Threonine
D006609:High Mobility Group Proteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D025741:HMGA1a Protein; D013912:Threonine; D004247:DNA; D006690:Bisbenzimidazole; D000409:Alanine