Structural and Mechanistic Analysis of (p)ppGpp Synthetases

The ability of microorganisms to survive under a large variety and rapidly changing environmental conditions is one of their most outstanding features and allowed them to establish within all niches of our planet. To do so, microorganisms have developed a mechanism called the stringent response (SR). The SR relies on the presence of the nucleotide second-messengers (p)ppGpp that contribute to reallocation of resources during stressful environmental conditions. Understanding the broad variety of adaptation processes mediated by the SR therefore necessitates to decipher the metabolism of (p)ppGpp. The stringent factor RelA was long thought to solely account for synthesis and degradation of (p)ppGpp. However, two additional (p)ppGpp synthesizing enzymes, SAS1 and SAS2, were discovered recently. This work presents an in-depth structural and mechanistic characterization of SAS1 and SAS2. Both proteins are subject to allosteric regulation allowing them to integrate different environmental stress stimuli into the framework of the SR. However, SAS1 and SAS2 also mediate adaptation of the microorganism in the absence of environmental stress stimuli, e.g. lack of nutrients. By this, they provide promising targets for the development of future antibiotics guided by the elucidation of their structure and mechanism present in this work. Analysis of (p)ppGpp effecting various cellular targets reveals that the SR confers adaptation processes in a wide intracellular concentration range. This sheds new light on the SR as a mechanism of gradual response to subtle changes in the environment rather than following an ‘all or nothing’ paradigm

Structural Basis for Regulation of the Opposing (p)ppGpp Synthetase and Hydrolase within the Stringent Response Orchestrator Rel

The stringent response enables metabolic adaptation of bacteria under stress conditions and is governed by RelA/SpoT Homolog (RSH)-type enzymes. Long RSH-type enzymes encompass an N-terminal domain (NTD) harboring the second messenger nucleotide (p)ppGpp hydrolase and synthetase activity and a stress-perceiving and regulatory C-terminal domain (CTD). CTD-mediated binding of Rel to stalled ribosomes boosts (p)ppGpp synthesis. However, how the opposing activities of the NTD are controlled in the absence of stress was poorly understood. Here, we demonstrate on the RSH-type protein Rel that the critical regulative elements reside within the TGS (ThrRS, GTPase, and SpoT) subdomain of the CTD, which associates to and represses the synthetase to concomitantly allow for activation of the hydrolase. Furthermore, we show that Rel forms homodimers, which appear to control the interaction with deacylated-tRNA, but not the enzymatic activity of Rel. Collectively, our study provides a detailed molecular view into the mechanism of stringent response repression in the absence of stress

Structural and Mechanistic Analysis of (p)ppGpp Synthetases

The ability of microorganisms to survive under a large variety and rapidly changing environmental conditions is one of their most outstanding features and allowed them to establish within all niches of our planet. To do so, microorganisms have developed a mechanism called the stringent response (SR). The SR relies on the presence of the nucleotide second-messengers (p)ppGpp that contribute to reallocation of resources during stressful environmental conditions. Understanding the broad variety of adaptation processes mediated by the SR therefore necessitates to decipher the metabolism of (p)ppGpp. The stringent factor RelA was long thought to solely account for synthesis and degradation of (p)ppGpp. However, two additional (p)ppGpp synthesizing enzymes, SAS1 and SAS2, were discovered recently. This work presents an in-depth structural and mechanistic characterization of SAS1 and SAS2. Both proteins are subject to allosteric regulation allowing them to integrate different environmental stress stimuli into the framework of the SR. However, SAS1 and SAS2 also mediate adaptation of the microorganism in the absence of environmental stress stimuli, e.g. lack of nutrients. By this, they provide promising targets for the development of future antibiotics guided by the elucidation of their structure and mechanism present in this work. Analysis of (p)ppGpp effecting various cellular targets reveals that the SR confers adaptation processes in a wide intracellular concentration range. This sheds new light on the SR as a mechanism of gradual response to subtle changes in the environment rather than following an ‘all or nothing’ paradigm

Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

Second messengers are free to diffuse through the cells and to activate all responsive elements. Cyclic di-GMP (c-di-GMP) signaling plays an important role in the determination of the life style transition between motility and sessility/biofilm formation but involves numerous distinct synthetases (diguanylate cyclases [DGCs]) or receptor pathways that appear to act in an independent manner. Using Bacillus subtilis as a model organism, we show that for two c-di-GMP pathways, DGCs and receptor molecules operate via direct interactions, where a synthesized dinucleotide appears to be directly used for the protein-protein interaction. We show that very few DGC molecules exist within cells; in the case of exopolysaccharide (EPS) formation via membrane protein DgcK, the DGC molecules act at a single site, setting up a single signaling pool within the cell membrane. Using single-molecule tracking, we show that the soluble DGC DgcP arrests at the cell membrane, interacting with its receptor, DgrA, which slows down motility. DgrA also directly binds to DgcK, showing that divergent as well as convergent modules exist in B. subtilis. Thus, local-pool signal transduction operates extremely efficiently and specifically.Bacillus subtilis contains two known cyclic di-GMP (c-di-GMP)-dependent receptors, YdaK and DgrA, as well as three diguanylate cyclases (DGCs): soluble DgcP and membrane-integral DgcK and DgcW. DgrA regulates motility, while YdaK is responsible for the formation of a putative exopolysaccharide, dependent on the activity of DgcK. Using single-molecule tracking, we show that a majority of DgcK molecules are statically positioned in the cell membrane but significantly less so in the absence of YdaK but more so upon overproduction of YdaK. The soluble domains of DgcK and of YdaK show a direct interaction in vitro, which depends on an intact I-site within the degenerated GGDEF domain of YdaK. These experiments suggest a direct handover of a second messenger at a single subcellular site. Interestingly, all three DGC proteins contribute toward downregulation of motility via the PilZ protein DgrA. Deletion of dgrA also affects the mobility of DgcK within the membrane and also that of DgcP, which arrests less often at the membrane in the absence of DgrA. Both, DgcK and DgcP interact with DgrA in vitro, showing that divergent as well as convergent direct connections exist between cyclases and their effector proteins. Automated determination of molecule numbers in live cells revealed that DgcK and DgcP are present at very low copy numbers of 6 or 25 per cell, respectively, such that for DgcK, a part of the cell population does not contain any DgcK molecule, rendering signaling via c-di-GMP extremely efficient

Molecular architecture of the DNA-binding sites of the P-loop ATPases MipZ and ParA from Caulobacter crescentus

The spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus is mediated by two different P-loop ATPases, ParA and MipZ. Both of these proteins form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization depends on their nucleotide-dependent cycling between a monomeric and a dimeric state and on the ability of the dimeric species to associate with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to comprehensively map the residues mediating the interactions of MipZ and ParA with DNA. We show that MipZ has non-specific DNA-binding activity that relies on an array of positively charged and hydrophobic residues lining both sides of the dimer interface. Extending our analysis to ParA, we find that the MipZ and ParA DNA-binding sites differ markedly in composition, although their relative positions on the dimer surface and their mode of DNA binding are conserved. In line with previous experimental work, bioinformatic analysis suggests that the same principles may apply to other members of the P-loop ATPase family. P-loop ATPases thus share common mechanistic features, although their functions have diverged considerably during the course of evolution

Cryo-EM structure of human eIF5A-DHS complex reveals the molecular basis of hypusination-associated neurodegenerative disorders

eIF5A is the only protein known to contain hypusine. Here, the authors present the cryoEM structure of the eIF5A-DHS complex and provide mechanistic insights to understand the deoxyhypusination reaction and hypusination-related neurodegeneration