title: Determinants of protein-protein recognition by four helix bundles: changing the dimerization specificity of Tet repressor.
abstract: Homo- and heterodimerization is essential for the activity of many proteins, particularly transcription factors. One widely distributed structural motif for protein recognition is the four helix bundle. To understand the molecular details determining specificity of subunit recognition in a dimer formed by a four helix bundle, we investigated Tet repressor (TetR) sequence variants TetR(B) and TetR(D), which do not form heterodimers. We used molecular modeling to identify residues with the potential to determine recognition of subunits. Directed mutagenesis of these residues in TetR(B) by the TetR(D) sequence resulted in chimeric TetR(B/D) repressors with new subunit recognition specificities. The single LS192 exchange in TetR(B/D)192 in the center of the helix bundle 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 subunit. 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 forms dimers with TetR(D) but not with TetR(B), indicating that four amino acid exchanges are sufficient to change subunit recognition. These results establish that targeted alterations in the structural complementarity of protein-protein interaction surfaces can be used to construct new recognition specificities. However, it is not sufficient to adjust the complementary residues since the surrounding amino acids contribute essentially to protein-protein recognition.
authors.affiliation: Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander Universität Erlangen-Nürnberg, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Schnappinger
authors.forename: P
authors.initials: P
authors.lastname: Schubert
authors.forename: K
authors.initials: K
authors.lastname: Pfleiderer
authors.forename: W
authors.initials: W
authors.lastname: Hillen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013752:Tetracycline
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline