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

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.