DETERMINANTS OF PROTEIN-PROTEIN RECOGNITION BY 4 HELIX BUNDLES - CHANGING THE DIMERIZATION SPECIFICITY OF TET REPRESSOR

Home-and heterodimerization is essential for the activity of many prot eins, particularly transcription factors, One widely distributed struc tural motif for protein recognition is the four helix bundle, To under stand the molecular details determining specificity of subunit recogni tion in a dimer formed by a four helix bundle, we investigated Tet rep ressor (TetR) sequence variants TetR(B) and TetR(D), which do not form heterodimers, We used molecular modeling to identify residues with th e potential to determine recognition of subunits, Directed mutagenesis of these residues in TetR(B) by the TetR(D) sequence resulted in chim eric TetR(B/D) repressors with new subunit recognition specificities, The single LS192 exchange in TetR(B/D)192 in the center of the helix b undle leads to a relaxed specificity since this variant dimerizes with TetR(B) and (D), To construct a variant with a new specificity it was not sufficient to mutate the contacting residue, F197, in the other s ubunit, Instead, it was necessary to exchange two more residues in the vicinity of F197 and S192, The resulting TetR(B/D)188,192,193,197 for ms dimers with TetR(D) but not with TetR(B), indicating that four amin o acid exchanges are sufficient to change subunit recognition, These r esults establish that targeted alterations in the structural complemen tarity of protein-protein interaction surfaces can be used to construc t new recognition specificities, However, it is not sufficient to adju st the complementary residues since the surrounding amino acids contri bute essentially to protein-protein recognition.