title: Computational design of a chain-specific tetracycline repressor heterodimer.
abstract: The specificity and selectivity of protein-protein interactions are of central importance for many biological processes, including signal transduction and transcription control. We used the in-house side-chain packing program MUMBO to computationally design a chain-specific heterodimeric variant of the bacterial transcription regulator tetracycline repressor (TetR), called T-A(A)B. Our goal was to engineer two different TetR chain variants, A and B, that no longer interact as AA or BB homodimers but selectively recombine to form heterodimers. Although 56 residues from each chain contribute to a dimer interface as large as 2200 Å(2) in wild-type TetR, the substitution of only three residues in one chain and two residues in a second chain sufficed for generating specificity in a T-A(A)B heterodimer variant. The design was corroborated in vivo by a cell-based transcription assay, and in vitro by CD spectroscopy and X-ray crystallography. Crystal structure analyses showed that while selectivity in the B chain is achieved entirely through van der Waals repulsion, the best selectivity in the A chain is obtained for the variant with the lowest number of atoms in the interface, thus possibly leading to underpacking of the dimer interface. This results in a marked decrease in thermal stability and a drastic reduction in the solubility of the T-A(A)A(A) homodimer in comparison to the designed T-A(A)B heterodimer variant.
authors.affiliation: Lehrstuhl für Biotechnik, Department of Biology, Friedrich-Alexander University Erlangen-Nuremberg, Im IZMP, Henkestr. 91, D-91052 Erlangen, Germany.
authors.forename: Martin T
authors.initials: MT
authors.lastname: Stiebritz
authors.forename: Stefanie
authors.initials: S
authors.lastname: Wengrzik
authors.forename: Doris L
authors.initials: DL
authors.lastname: Klein
authors.forename: Jan Paul
authors.initials: JP
authors.lastname: Richter
authors.forename: Anna
authors.initials: A
authors.lastname: Srebrzynski
authors.forename: Sigrid
authors.initials: S
authors.lastname: Weiler
authors.forename: Yves A
authors.initials: YA
authors.lastname: Muller
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D019295:Computational Biology; D018360:Crystallography, X-Ray; D019281:Dimerization; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline