C-di-GMP Synthesis: Structural Aspects of Evolution, Catalysis and Regulation

Cellular levels of the second messenger cyclic di-guanosine monophosphate (c-di-GMP) are determined by the antagonistic activities of diguanylate cyclases and specific phosphodiesterases. In a given bacterial organism, there are often multiple variants of the two enzymes, which are tightly regulated by a variety of external and internal cues due to the presence of specialized sensory or regulatory domains. Dependent on the second messenger level, specific c-di-GMP receptors then control fundamental cellular processes, such as bacterial life style, biofilm formation, and cell cycle control. Here, I review the large body of data on structure-function relationships in diguanylate cyclases. Although the catalytic GGDEF domain is related to the respective domain of adenylate cyclases, the catalyzed intermolecular condensation reaction of two GTP molecules requires the formation of a competent GGDEF dimer with the two substrate molecules juxtaposed. This prerequisite appears to constitute the basis for GGDEF regulation with signal-induced changes within the homotypic dimer of the input domain (PAS, GAF, HAMP, etc.), which are structurally coupled with the arrangement of the GGDEF domains via a rigid coiled-coil linker. Alternatively, phosphorylation of a Rec input domain can drive GGDEF dimerization. Both mechanisms allow modular combination of input and output function that appears advantageous for evolution and rationalizes the striking similarities in domain architecture found in diguanylate cyclases and histidine kinases

Efficient Enzymatic Production of the Bacterial Second Messenger c-di-GMP by the Diguanylate Cyclase YdeH from E. coli

Cyclic di-GMP (c-di-GMP) is an almost universal bacterial second messenger involved in the regulation of cell surface-associated traits and the persistence of infections. GGDEF and EAL domain-containing proteins catalyse c-di-GMP synthesis and degradation, respectively. We report the enzymatic large-scale synthesis of c-di-GMP, making use of the GGDEF domain-containing protein YdeH from Escherichia coli. Overexpression and purification of YdeH have been established, and the conditions for c-di-GMP synthesis were optimised. In contrast to the chemical synthesis of c-di-GMP, enzymatic c-di-GMP production is a one-step reaction that can easily be performed with the equipment of a standard biochemical lab. The protocol allows the production of milligram amounts of c-di-GMP within 1day and paves the way for extensive biochemical and biophysical studies on c-di-GMP-mediated processe

Activation mechanism of a small prototypic Rec-GGDEF diguanylate cyclase

Diguanylate cyclases (DGCs) 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. 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 DGCs. Full-length crystal structures revealed that BeF 3 - 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 DGCs with N-terminal coiled-coil linkers

Cyclic di-GMP mediates a histidine kinase/phosphatase switch by noncovalent domain cross-linking

Histidine kinases are key components of regulatory networks in bacteria. Although many of these enzymes are bifunctional, mediating both phosphorylation and dephosphorylation of downstream targets, the molecular details of this central regulatory switch are unclear. We showed recently that the universal second messenger cyclic di-guanosine monophosphate (c-di-GMP) drives Caulobacter crescentus cell cycle progression by forcing the cell cycle kinase CckA from its default kinase into phosphatase mode. We use a combination of structure determination, modeling, and functional analysis to demonstrate that c-di-GMP reciprocally regulates the two antagonistic CckA activities through noncovalent cross-linking of the catalytic domain with the dimerization histidine phosphotransfer (DHp) domain. We demonstrate that both c-di-GMP and ADP (adenosine diphosphate) promote phosphatase activity and propose that c-di-GMP stabilizes the ADP-bound quaternary structure, which allows the receiver domain to access the dimeric DHp stem for dephosphorylation. In silico analyses predict that c-di-GMP control is widespread among bacterial histidine kinases, arguing that it can replace or modulate canonical transmembrane signaling

Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification

Filamentation induced by cyclic AMP (FIC)-domain enzymes catalyze adenylylation or other posttranslational modifications of target proteins to control their function. Recently, we have shown that Fic enzymes are autoinhibited by an α-helix (αinh) that partly obstructs the active site. For the single-domain class III Fic proteins, the αinh is located at the C terminus and its deletion relieves autoinhibition. However, it has remained unclear how activation occurs naturally. Here, we show by structural, biophysical, and enzymatic analyses combined with in vivo data that the class III Fic protein NmFic from Neisseria meningitidis gets autoadenylylated in cis, thereby autonomously relieving autoinhibition and thus allowing subsequent adenylylation of its target, the DNA gyrase subunit GyrB. Furthermore, we show that NmFic activation is antagonized by tetramerization. The combination of autoadenylylation and tetramerization results in nonmonotonic concentration dependence of NmFic activity and a pronounced lag phase in the progress of target adenylylation. Bioinformatic analyses indicate that this elaborate dual-control mechanism is conserved throughout class III Fic proteins

Structural features discriminating hybrid histidine kinase Rec domains from response regulator homologs

In two-component systems, the information gathered by histidine kinases (HKs) are relayed to cognate response regulators (RRs). Thereby, the phosphoryl group of the auto-phosphorylated HK is transferred to the receiver (Rec) domain of the RR to allosterically activate its effector domain. In contrast, multi-step phosphorelays comprise at least one additional Rec (Rec; inter; ) domain that is typically part of the HK and acts as an intermediary for phosphoryl-shuttling. While RR Rec domains have been studied extensively, little is known about discriminating features of Rec; inter; domains. Here we study the Rec; inter; domain of the hybrid HK CckA by X-ray crystallography and NMR spectroscopy. Strikingly, all active site residues of the canonical Rec-fold are pre-arranged for phosphoryl-binding and BeF; 3; -; binding does not alter secondary or quaternary structure, indicating the absence of allosteric changes, the hallmark of RRs. Based on sequence-covariation and modeling, we analyze the intra-molecular DHp/Rec association in hybrid HKs

Imaging the Electrostatic Potential of Transmembrane Channels: Atomic Probe Microscopy of OmpF Porin

This is the published version. Copyright 2002 by Elsevier.The atomic force microscope (AFM) was used to image native OmpF porin and to detect the electrostatic potential generated by the protein. To this end the OmpF porin trimers from Escherichia coli was reproducibly imaged at a lateral resolution of ∼0.5 nm and a vertical resolution of ∼0.1 nm at variable electrolyte concentrations of the buffer solution. At low electrolyte concentrations the charged AFM probe not only contoured structural details of the membrane protein surface but also interacted with local electrostatic potentials. Differences measured between topographs recorded at variable ionic strength allowed mapping of the electrostatic potential of OmpF porin. The potential map acquired by AFM showed qualitative agreement with continuum electrostatic calculations based on the atomic OmpF porin embedded in a lipid bilayer at the same electrolyte concentrations. Numerical simulations of the experimental conditions showed the measurements to be reproduced quantitatively when the AFM probe was included in the calculations. This method opens a novel avenue to determine the electrostatic potential of native protein surfaces at a lateral resolution better than 1 nm and a vertical resolution of ∼0.1 nm

Intercepting second-messenger signaling by rationally designed peptides sequestering c-di-GMP

The bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a wide range of cellular functions from biofilm formation to growth and survival. Targeting a second-messenger network is challenging because the system involves a multitude of components with often overlapping functions. Here, we present a strategy to intercept c-di-GMP signaling pathways by directly targeting the second messenger. For this, we developed a c-di-GMP-sequestering peptide (CSP) that was derived from a CheY-like c-di-GMP effector protein. CSP binds c-di-GMP with submicromolar affinity. The elucidation of the CSP⋅c-di-GMP complex structure by NMR identified a linear c-di-GMP-binding motif, in which a self-intercalated c-di-GMP dimer is tightly bound by a network of H bonds and π-stacking interactions involving arginine and aromatic residues. Structure-based mutagenesis yielded a variant with considerably higher, low-nanomolar affinity, which subsequently was shortened to 19 residues with almost uncompromised affinity. We demonstrate that endogenously expressed CSP intercepts c-di-GMP signaling and effectively inhibits biofilm formation in Pseudomonas aeruginosa , the most widely used model for serious biofilm-associated medical implications

Structural basis for selective targeting of Rac subfamily GTPases by a bacterial effector protein

Ras-homology (Rho) family GTPases are conserved molecular switches controlling fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, molecular mechanisms of target selectivity have remained elusive. Here, we report a bacterial effector protein that targets all four Rac subfamily members of Rho family GTPases, but none of the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 facilitate modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural paradigm for target selectivity towards Rac subfamily GTPases and provides a highly selective tool for their functional analysis

An Alternative Active Site Architecture for O2 Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum

Sulfoxide synthases are nonheme iron enzymes that catalyze oxidative carbon-sulfur bond formation between cysteine derivatives and N-α-trimethylhistidine as a key step in the biosynthesis of thiohistidines. The complex catalytic mechanism of this enzyme reaction has emerged as the controversial subject of several biochemical and computational studies. These studies all used the structure of the γ-glutamyl cysteine utilizing sulfoxide synthase, MthEgtB from Mycobacterium thermophilum (EC 1.14.99.50), as a structural basis. To provide an alternative model system, we have solved the crystal structure of CthEgtB from Chloracidobacterium thermophilum (EC 1.14.99.51) that utilizes cysteine as a sulfur donor. This structure reveals a completely different configuration of active site residues that are involved in oxygen binding and activation. Furthermore, comparison of the two EgtB structures enables a classification of all ergothioneine biosynthetic EgtBs into five subtypes, each characterized by unique active-site features. This active site diversity provides an excellent platform to examine the catalytic mechanism of sulfoxide synthases by comparative enzymology, but also raises the question as to why so many different solutions to the same biosynthetic problem have emerged