Abstract
Diguanylate cyclases synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown. As part of two-component systems, they are activated by cognate histidine kinases that phosphorylate their Rec input domains. DgcR from Leptospira biflexa is a constitutively dimeric prototype of this class of diguanylate cyclases. Full-length crystal structures reveal that BeF3- pseudo-phosphorylation induces a relative rotation of two rigid halves in the Rec domain. This is coupled to a reorganisation of the dimeric structure with concomitant switching of the coiled-coil linker to an alternative heptad register. Finally, the activated register allows the two substrate-loaded GGDEF domains, which are linked to the end of the coiled-coil via a localised hinge, to move into a catalytically competent dimeric arrangement. Bioinformatic analyses suggest that the binary register switch mechanism is utilised by many diguanylate cyclases with N-terminal coiled-coil linkers.
Highlights
Diguanylate cyclases synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown
Common input domains are GAF6 and PAS7 that can recognise a variety of molecules, and response regulator receiver domains (Rec)[8], which as part of two-component systems are phosphorylated by cognate histidine kinases (HKs)[9]
To reveal the structural changes accompanying the activation of Rec-GGDEF diguanylate cyclases (DGCs) we determined the full-length crystal structures of DgcR in native and pseudo-phosphorylated (BeF3- modified) state
Summary
Diguanylate cyclases synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown. Dozens of these enzymes can be encoded by one single genome with each of the proteins containing distinct sensory input domains that can sense/receive diverse signals like O2, light and metals[3,4,5] This allows bacteria to detect intracellular and environmental cues and respond promptly by adjusting c-di-GMP levels, which will be detected by specific receptors. We proposed earlier that the constituting helices could change their crossing angle and/or azimuthal orientation to allow or prevent productive encounter of the two GGDEF domains (chopstick model[10]) This mechanism would be a generalisation of the scissors with fixed pivot blades model ascribed to histidine kinases signalling[15]. LEPBI_RS18680 from Leptospira biflexa, hereafter called DgcR (diguanylate cyclase controlled by Rec), is a Rec-GGDEF protein with a short domain linker (Fig. 1a), making this enzyme attractive for studying its conformational states by crystallography
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