STRUCTURE AND MECHANISM OF ACTION OF THE LYSR-TYPE TRANSCRIPTIONAL REGULATOR, CYSB
K.H.G. Verschueren, C. Addy, G.N. Murshudov, E.J. Dodson, A.J. Wilkinson
Protein Structure Research Group, University of York, Heslington, York YO1 5DD, UK
The LysR-type transcriptional regulators (LTTRs) constitute a large family of bacterial gene activator proteins which control the expression of genes associated with a multitude of cellular processes. These processes are highly diverse, ranging from amino acid biosynthesis, CO2 fixation, ion transport, antibiotic resistance, initiation of nodulation, chromosomal replication and control of virulence [1]. After the two-component response systems, the LTTRs which currently comprises over 100 representatives, are the second largest family of transcriptional activators in bacteria. In vivo, they act either as tetramers or dimers of identical polypeptides, 270-330 amino acid resiues in length. These share stretches of sequence similarity over ~280 residues with the strongest conservation of sequence at their N-terminal 66 residues which include a putative helix-turn-helix motif believed to play a role in DNA binding. Most of these proteins have a dual regulatory function, acting as transcriptional activators at one or several loci whilst negatively regulating transcription of their own gene.
Cysteine biosynthesis in Gram-negative bacteria requires the products of a total of 22 cys genes, of which at least 14 are coordinately regulated and collectively referred to as the cysteine regulon [2]. This regulation is dependent on the activity of the LTTR protein CysB, the presence of elevated concentrations of inducer N-acetylserine (NAS), and sulphur limitation. CysB is a tetrameric protein of identical 36 kDa subunits (324 residues) which binds upstream of the -35 region of a number of cys promoters and, in the presence of NAS activates transcription. CysB also binds in a region which overlaps the RNA polymerase binding site of its own promoter where it acts as a repressor of transcription. Repression at this site is lifted by NAS. Sulphide and thiosulphate act as antiinducers reversing the effects of Nacetylserine. The interactions of CysB with cys promoters are extremely complex due to the existence of multiple overlapping binding sites, the absence of well-conserved promoter sequence, and the involvement of DNA bending.
The crystal structure of a dimeric chymotryptic fragment of CysB from Klebsiella aerogenes, residues 88324, containing the cofactorbinding domain has been elucidated and refined to a resolution of 1.8A using a combination of MIR (multiple isomorphous replacement) techniques and multicrystal averaging [3]. This structure provides the first three-dimensional insight into a protein belonging to the LTTRs giving the results a wider significance. CysB(88324) still binds the cofactor, Nacetylserine, although it lacks the N-terminal DNA binding determinants. The core of the structure consists of two á/â domains connected by a short twostranded hinge region. A deep cavity is formed between the two domains. This cavity most likely represents the cofactor binding site based on the structural similarity of CysB(88324) to periplasmic receptor proteins in which the corresponding cavity represents the substrate binding site. Surprisingly, the cleft in CysB(88324) is occupied by a sulphate anion. Unlike full-length CysB which is active as a tetramer, this fragment is a dimer.
The cofactor binding domain of the monomer also closely resembles those of the IPTG and hypoxanthine binding domains of lac repressor and PurR respectively. However the subunit organisation of the two molecules in the CysB(88324) dimer is quite different to that in lac repressor and PurR. Whereas in the latter the twofold axis of symmetry relating monomers in the dimer is parallel to the long axis of the cofactor binding domain, in CysB(88324) it is perpendicular to this axis. This is expected to position the Nterminal DNA binding domains at opposite ends of the molecule perhaps accounting for the unusually large DNA footprints produced by CysB.