Allosteric control of the c-di-GMP metabolism: insights from RmcA hybrid protein

Biofilm is a microbial community whose formation is regulated by the dinucleotide cyclic-di GMP. The GGDEF diguanylate cyclases and EAL or HD-GYP phosphodiesterases control the balance of c-di-GMP. Most of these proteins have N-terminal sensing domains indicating that their activities are modulated by cellular and environmental stimuli. Moreover there are proteins bearing both the GGDEF and EAL or HD-GYP domains named hybrid proteins. The cross-talk between these two domains and with other regulatory domains is crucial to understand the output and the regulation of hybrid proteins. Given that nutrients are among the major driving forces guiding the change of c-diGMP levels, the characterization of the mechanistic details linking nutrients sensing to c-di-GMP homeostasis is very interesting and up to now poorly characterized. The aim of study was to identify a putative protein in P. aeruginosa able to link the nutrient state to c-di-GMP signalling. In particular my project has been focused on the characterization of the Hybrid Protein RmcA (Redox modulator of c-di-GMP A) from Pseudomonas aeruginosa. RmcA is a multidomain transmembrane protein carrying a periplasmic Venus Fly Trap (VFT) sensory domain, a transmembrane helix, three Per-Arnt-Sim (PAS) domain and one light, oxygen, or voltage domain (LOV) and finally the GGDEF-EAL domains.Few structural data are available on Hybrid Protein family of enzymes and these data indicate that the output of these proteins is regulated by a reciprocal allosteric control between the two GGDEF and EAL domains occuring via inter-domain interactions and ligand-induced conformationals changes. In addition, the final output is difficult to predict since it depends also on the interaction between the two domains and the upstream sensing/regulatory ones. During the enzymatic characterization of RmcA, we observed alternative and novel reactivity of the GGDEF domain which has been analysed more deeply, also in comparison with other GGDEFcontaining enzymes. We found that DUAL protein showed an unusual catalytic activity converting GTP to GMP. We therefore investigated whether this unexpected feature is a common feature of different GGDEF containing proteins, including both DGCs and hybrid proteins. This side project has been useful for the overall study presented in this PhD thesis, since it allowed us to establish the best experimental conditions to study this domain.