Structural elucidation of the multidomain response regulator PleD using X-ray crystallography

Abstract

c-diGMP (bis-(3!5)-cyclic di-guanosine monophosphate) is used extensively in bacteria to control biofilm formation and is lately postulated as a novel secondary messenger. Little is known about the signalling process, nor the control, of this dinucleotide. It is clear, however, that its synthesis is catalysed by the DGC (diguanylate cyclase) domain that contains a conserved GG(D/E)EF sequence motif. Despite its high abundance in bacteria, the structure was until now unknown. The PleD protein from Caulobacter crescentus contains a C-terminal DGC domain, preceded by the input domain D1 and the adaptor domain D2. PleD is a response regulator from the two-component signalling system. The output DGC response relies phosphorylation at the N-terminal D1 input domain. Therefore, the control of c-diGMP signal can be revealed in this multi-domain protein. The objectives of my PhD work are to (1) reveal the structure of DGC domain, (2) understand the catalytic mechanism of DGC, and (3) understand the regulation of the DGC response through the structure of PleD. The crystal structure of PleD has been solved in complex with c-diGMP to 2.7 °A. The fold of the DGC domain is similar to adenylate cyclase, but the proposed nucleotide binding mode is substantially different. The crystal packing has suggested that two DGC domains align in a two-fold symmetric way to catalyse c-diGMP synthesis. Hence, PleD is active as a dimer using D1 and D2 domains for dimerisation. The dimer formation is probably caused by phosphorylation at the D1 domain. In addition, the structure shows that two intercalated products bind at the D2-DGC domain interface. Such binding is thought to serve an allosteric purpose by immobilising DGC domain movements and prevent them from forming the active site. This thesis reports the crystal structure of PleD in complex with cdiGMP, and discusses the implications of the structure on DGC catalysis and on activation and inhibition regulation of DGC activity in PleD. In addition, the thesis describes the preparative investigations and characteri sation that have led to structure determination of PleD. These include the design and screening of PleD constructs, the establishment and optimisation of expression and purification, protein characterisation, crystallisation optimisation, and diffraction data collection.