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 Emilia Stec 1 , Malgorzata Witkowska-Zimny 2 , Monika M. Hryniewicz 2 ⁎ Piotr Neumann 3 , Anthony J. Wilkinson 4 , Andrzej M. Brzozowski 4 Chandra S. Verma 5 , Jolanta Zaim 6 , Stanislaw Wysocki 1 and Grzegorz D. Bujacz 1 ⁎ 1 Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Poland 2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland 3 Faculty of Chemistry, Nicolaus Copernicus University of Torun, Gagarina 7, 87-100 Torun, Poland 4 Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, UK 5 Bioinformatics Institute, Department of Biological Sciences, National University of Singapore, 30 Biopolis Way, #07-01 Matrix, Singapore 138671, Singapore 6 Systems Biology and Bioinformatics Research Group Maltanska 4/37 02-761 Warsaw, Poland 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. © 2006 Elsevier Ltd. All rights reserved. *Corresponding authors Keywords: E. coli; Cbl regulator; LysR family; crystal structure; cofactor-binding Introduction The process of “sulphur assimilation” from inorganic sulphate through the cysteine biosynthetic pathway is common to bacteria, fungi, and plants and proceeds by essentially similar steps of reduc- tion of S 6+ to S 2− followed by the incorporation of Abbreviations used: PBP, periplasmic-binding protein; LTTR, LysR-type transcriptional regulators; RD, regulatory domain; APS, adenosine phosphosulphate; NAS, N-acetyl-L-serine; DBD, DNA-binding domain; OAS, O-acetyl-L-serine. E-mail addresses of the corresponding authors: monikah@ibb.waw.pl; gdbujacz@p.lodz.pl doi:10.1016/j.jmb.2006.06.033 J. Mol. Biol. (2006) 364, 309–322 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.