2uye
From Proteopedia
DOUBLE MUTANT Y110S,F111V DNTR FROM BURKHOLDERIA SP. STRAIN DNT IN COMPLEX WITH THIOCYANATE
Overview
DntR is a bacterial transcription factor that has been isolated from Burkholderia species that are able to degrade the nitro-aromatic compound 2,4-dinitrotoluene. We recently solved the X-ray crystal structure of DntR, which suggested a putative location of an inducer-binding cavity (IBC). In this study, we constructed mutants of DntR in which residues lining the proposed IBC were modified in order to identify the structural elements involved in inducer binding, to modulate the inducer binding specificity, and to investigate the mechanism of transcriptional regulation by DntR. The transcriptional activation of the reporter gene gfp induced by the wild-type and mutant DntRs was monitored by analysing whole-cell fluorescence using flow-cytometry after addition of a number of potential inducer compounds. Three of the mutant proteins (F111L; F111V/H169V and Y110S/F111V) were purified and the binding constants for several of the potential inducers to these mutants were estimated. Furthermore, crystal structures of the F111L and Y110S/F111V mutant proteins were solved and used to explain changes in the inducer binding specificity at an atomic level. A comparison of the inducing capability in the whole-cell system and binding constants for a number of potential inducers suggests a mechanism where binding of an inducer molecule is not the sole requirement for transcriptional activation. In addition, specific interactions between DntR and the inducer molecule resulting in a conformational change of the protein are needed.
About this Structure
2UYE is a Single protein structure of sequence from Burkholderia cepacia. Full crystallographic information is available from OCA.
Reference
In vivo and in vitro investigation of transcriptional regulation by DntR., Lonneborg R, Smirnova I, Dian C, Leonard GA, Brzezinski P, J Mol Biol. 2007 Sep 21;372(3):571-82. Epub 2007 Jul 3. PMID:17681542 Page seeded by OCA on Sun May 4 17:48:49 2008
