THE STRUCTURE OF THE COFACTOR-BINDING FRAGMENT OF THE LYSR FAMILY MEMBER, CYSB - A FAMILIAR FOLD WITH A SURPRISING SUBUNIT ARRANGEMENT

Background: CysB is a tetrameric protein of identical subunits (M-r = 36,000) which controls the expression of genes associated with the bio synthesis of cysteine in bacteria. CysB is both an activator and a rep ressor of transcription whose activity is responsive to the inducer N- acetylserine; thiosulphate and sulphide act as anti-inducers. CysB is a member of the LysR family of prokaryotic transcriptional regulatory proteins which share sequence similarities over similar to 280 residue s including a putative helix-turn-helix DNA-binding motif at their N t erminus. The aims of the present study were to explore further the com plex molecular biology and curious ligand binding properties of CysB a nd to provide structural insights into the LysR family of proteins. Re sults: The crystal structure of a dimeric chymotryptic fragment of Kle bsiella aerogenes CysB comprising residues 88-324, has been solved by multiple isomorphous replacement and multi-crystal averaging and refin ed against data extending to 1.8 Angstrom resolution. The protein comp rises two alpha/beta domains (I and II) connected by two short segment s of polypeptide. The two domains enclose a cavity lined by polar side chains, including those of two residues whose mutation is associated w ith constitutive expression of the cysteine regulon. A sulphate anion and a number of well ordered water molecules have been modelled into d iscrete electron-density peaks within this cavity. In the dimer, stran ds beta(B) from domain I and strands beta(G) from domain II come toget her so that a pair of antiparallel symmetry-related 11-stranded twiste d beta-pleated sheets is formed. Conclusions: The overall structure of CysB(88-324) is strikingly similar to those of the periplasmic substr ate-binding proteins. A similar fold has also been observed in the cof actor-binding domain of Lac repressor, implying a structural relations hip between the Lac repressor and LysR families of proteins. In contra st to Lac repressor, in CysB the twofold axis of symmetry that relates the monomers in the dimer is perpendicular rather than parallel to th e long axis of the cofactor-binding domain. This seems likely to place the DNA-binding domains at opposite extremes of the molecule possibly accounting for CysB's extended DNA footprints.