Structural basis of the sulphate starvation response in E. coli: crystal structure and mutational analysis of the cofactor-binding domain of the Cbl transcriptional regulator.
Cbl is a member of the large family of LysR-type transcriptional regulators (LTTRs) common in bacteria and found also in Archaea and algal chloroplasts. The function of Cbl is required in Escherichia coli for expression of sulphate starvation-inducible (ssi) genes, associated with the biosynthesis of cysteine from organic sulphur sources (sulphonates). Here, we report the crystal structure of the cofactor-binding domain of Cbl (c-Cbl) from E. coli. The overall fold of c-Cbl is very similar to the regulatory domain (RD) of another LysR family member, CysB. The RD is composed of two subdomains enclosing a cavity, which is expected to bind effector molecules. We have constructed and analysed several full-length Cbl variants bearing single residue substitutions in the RD that affect cofactor responses. Using in vivo and in vitro transcription assays, we demonstrate that pssuE, a Cbl responsive promoter, is down-regulated not only by the cofactor, adenosine phosphosulphate (APS), but also by thiosulphate, and, that the same RD determinants are important for the response to both cofactors. We also demonstrate the effects of selected site-directed mutations on Cbl oligomerization and discuss these in the context of the structure. Based on the crystal structure and molecular modelling, we propose a model for the interaction of Cbl with adenosine phosphosulphate.
Stec
Emilia
E
Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Poland.
Witkowska-Zimny
Malgorzata
M
Hryniewicz
Monika M
MM
Neumann
Piotr
P
Wilkinson
Anthony J
AJ
Brzozowski
Andrzej M
AM
Verma
Chandra S
CS
Zaim
Jolanta
J
Wysocki
Stanislaw
S
Bujacz
Grzegorz D
GD
D000250:Adenosine Phosphosulfate / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015536:Down-Regulation; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013885:Thiosulfates / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
C097229:Cbl protein, E coli; D029968:Escherichia coli Proteins; D013885:Thiosulfates; D014157:Transcription Factors; D000250:Adenosine Phosphosulfate