2XGC: Crystal Structure Of A Designed Heterodimeric Variant T-A(I) B Of The Tetracycline Repressor

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 A(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.
PDB ID: 2XGCDownload
MMDB ID: 84971
PDB Deposition Date: 2010/6/3
Updated in MMDB: 2010/10
Experimental Method:
x-ray diffraction
Resolution: 2.15  Å
Source Organism:
Photobacterium damselae subsp. piscicida
Similar Structures:
Biological Unit for 2XGC: dimeric; determined by author and by software (PISA)
Molecular Components in 2XGC
Label Count Molecule
Proteins (2 molecules)
Tetracycline Repressor Protein Class B From Transposon Tn10, Tetracycline Repressor Protein Class D
Molecule annotation
* Click molecule labels to explore molecular sequence information.

Citing MMDB