Summary
The alarmones (p)ppGpp are important second messengers that orchestrate pleiotropic adaptations of bacteria and plant chloroplasts in response to starvation and stress. Here, we review our ...structural and mechanistic knowledge on (p)ppGpp metabolism including their synthesis, degradation and interconversion by a highly diverse set of enzymes. Increasing structural information shows how (p)ppGpp interacts with an incredibly diverse set of different targets that are essential for replication, transcription, translation, ribosome assembly and metabolism. This raises the question how the chemically rather simple (p)ppGpp is able to interact with these different targets? Structural analysis shows that the diversity of (p)ppGpp interaction with cellular targets critically relies on the conformational flexibility of the 3′ and 5′ phosphate moieties allowing alarmones to efficiently modulate the activity of target structures in a broad concentration range. Current approaches in the design of (p)ppGpp‐analogs as future antibiotics might be aided by the comprehension of conformational flexibility exhibited by the magic dancers (p)ppGpp.
The alarmones (p)ppGpp are essential second messengers conserved among bacteria and the plant chloroplasts. This review discusses the molecular plasticity of enzymes and targets involved in (p)ppGpp metabolism and regulation, respectively.
When bacteria experience growth-limiting environmental conditions, the synthesis of the hyperphosphorylated guanosine derivatives (p)ppGpp is induced by enzymes of the RelA/SpoT homology (RSH)-type ...protein family. High levels of (p)ppGpp induce a process called "stringent response", a major cellular reprogramming during which ribosomal RNA (rRNA) and transfer RNA (tRNA) synthesis is downregulated, stress-related genes upregulated, messenger RNA (mRNA) stability and translation altered, and allocation of scarce resources optimized. The (p)ppGpp-mediated stringent response is thus often regarded as an all-or-nothing paradigm induced by stress. Over the past decades, several binding partners of (p)ppGpp have been uncovered displaying dissociation constants from below one micromolar to more than one millimolar and thus coincide with the accepted intracellular concentrations of (p)ppGpp under non-stringent (basal levels) and stringent conditions. This suggests that the ability of (p)ppGpp to modulate target proteins or processes would be better characterized as an unceasing continuum over a concentration range instead of being an abrupt switch of biochemical processes under specific conditions. We analyzed the reported binding affinities of (p)ppGpp targets and depicted a scheme for prioritization of modulation by (p)ppGpp. In this ranking, many enzymes of e.g., nucleotide metabolism are among the first targets to be affected by rising (p)ppGpp while more fundamental processes such as DNA replication are among the last. This preference should be part of (p)ppGpp's "magic" in the adaptation of microorganisms while still maintaining their potential for outgrowth once a stressful condition is overcome.
In eukaryotes, most secretory and membrane proteins are targeted by an N‐terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein‐conducting channel ...(PCC). In the post‐translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre‐opened state. Here, we present a cryo‐EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N‐terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C‐terminus of Sec63. Together, we obtained a near‐complete and integrated model of the active Sec complex.
SYNOPSIS
The overall architecture of the eukaryotic SecY/Sec61 channel for post‐translational protein translocation, and how it recognizes N‐terminal signal sequences in transported proteins, remain unclear. Here, structural analyses reveal the molecular architecture of the engaged yeast Sec complex, which binds the signal sequence in a unique open conformation.
Cryo‐EM structures of the heptameric Sec complex reveal the conformational transition from the partially open apo‐state to the fully open signal sequence‐bound state.
The signal sequence binds the lateral gate of the Sec61 complex, which is stabilized by the transmembrane helices of the Sec62 subunit.
The X‐ray structure of the N‐terminal cytoplasmic domain of Sec62 reveals a novel fold that is coordinated by the phosphorylated C‐terminal peptide of the Sec63 subunit.
Cryo‐electron microscopy and X‐ray crystallography studies reveal the molecular architecture of the engaged eukaryotic heptameric Sec complex, binding the protein signal sequence in an open conformation.
Summary
Cryptochrome (cry) blue light photoreceptors have important roles in the regulation of plant development. Their photocycle includes redox changes of their flavin adenine dinucleotide (FAD) ...chromophore, which is fully oxidised in the dark state and semi‐reduced in the signalling‐active lit state. The two Arabidopsis thaliana cryptochromes, cry1 and cry2, and the plant‐type cryptochrome CPH1 from Chlamydomonas rheinhardtii bind ATP and other nucleotides. Binding of ATP affects the photocycle of these photoreceptors and causes structural alterations. However, the exact regions that undergo structural changes have not been defined, and most importantly it is not known whether ATP binding affects the biological activity of these photoreceptors in planta. Here we present studies on the effect of ATP on Arabidopsis cry2. Recombinant cry2 protein showed a high affinity for ATP (KD of 1.09 ± 0.48 μm). Binding of ATP and other adenines promoted photoreduction of the FAD chromophore in vitro and caused structural changes, particularly in α‐helix 21 which links the photosensory domain with the C‐terminal extension. The constructed cry2Y399A mutant was unable to bind ATP and did not show enhancement of photoreduction by ATP. When this mutant gene was expressed in Arabidopsis null cry2 mutant plants it retained some biological activity, which was, however, lower than that of the wild type. Our results indicate that binding of ATP to cry2, and most likely to other plant‐type cryptochromes, is not essential but boosts the formation of the signalling state and biological activity.
Significance Statement
Binding of ATP by plant cryptochromes enhances signalling state formation, but how this affects the biological activity of these blue light photoreceptors was unknown. Here we show that a mutant of Arabidopsis thaliana cry2, which is unable to bind ATP, is still biologically active but to a smaller degree than the wild‐type photoreceptor. Thus, binding of ATP to cry2 in vivo is relevant for its biological function.
The evolution of ribulose-1,5-bisphosphate carboxylase/oxygenases (Rubiscos) that discriminate strongly between their substrate carbon dioxide and the undesired side substrate dioxygen was an ...important event for photosynthetic organisms adapting to an oxygenated environment. We use ancestral sequence reconstruction to recapitulate this event. We show that Rubisco increased its specificity and carboxylation efficiency through the gain of an accessory subunit before atmospheric oxygen was present. Using structural and biochemical approaches, we retrace how this subunit was gained and became essential. Our work illuminates the emergence of an adaptation to rising ambient oxygen levels, provides a template for investigating the function of interactions that have remained elusive because of their essentiality, and sheds light on the determinants of specificity in Rubisco.
Origins of Rubisco’s small subunit sidekick
Maintaining the correct activity, specificity, and stability is a challenge faced by metabolic enzymes that is often solved by forming multimeric complexes. The large, catalytic subunit of form I Rubisco, the carbon-fixing enzyme found in aerobic photosynthetic organisms such as plants and cyanobacteria and in some nonphotosynthetic bacteria, makes such a complex with an essential small subunit. Using ancestral sequence reconstruction, Schulz
et al
. investigated how recruiting this small subunit contributed to Rubisco evolution and, in particular, its specificity for carbon dioxide (see the Perspective by Sharwood). Enzyme activity assays and macromolecular structures revealed that the small subunit quickly became essential and opened the door to broader functional changes within the large subunit, including increased activity and specificity for carbon dioxide. —MAF
Ancestral sequence reconstruction reveals how recruiting a new subunit primes form I Rubiscos for rising atmospheric oxygen.
In quorum sensing, bacteria secrete or release small molecules into the environment that, once they reach a certain threshold, trigger a behavioural change in the population. As the concentration of ...these so-called autoinducers is supposed to reflect population density, they were originally assumed to be continuously produced by all cells in a population. However, here we show that in the α-proteobacterium Sinorhizobium meliloti expression of the autoinducer synthase gene is realized in asynchronous stochastic pulses that result from scarcity and, presumably, low binding affinity of the key activator. Physiological cues modulate pulse frequency, and pulse frequency in turn modulates the velocity with which autoinducer levels in the environment reach the threshold to trigger the quorum sensing response. We therefore propose that frequency-modulated pulsing in S. meliloti represents the molecular mechanism for a collective decision-making process in which each cell's physiological state and need for behavioural adaptation is encoded in the pulse frequency with which it expresses the autoinducer synthase gene; the pulse frequencies of all members of the population are then integrated in the common pool of autoinducers, and only once this vote crosses the threshold, the response behaviour is initiated.
The guanosine nucleotide‐based second messengers ppGpp and pppGpp (collectively: (p)ppGpp) enable adaptation of microorganisms to environmental changes and stress conditions. In contrast, the closely ...related adenosine nucleotides (p)ppApp are involved in type VI secretion system (T6SS)‐mediated killing during bacterial competition. Long RelA‐SpoT Homolog (RSH) enzymes regulate synthesis and degradation of (p)ppGpp (and potentially also (p)ppApp) through their synthetase and hydrolase domains, respectively. Small alarmone hydrolases (SAH) that consist of only a hydrolase domain are found in a variety of bacterial species, including the opportunistic human pathogen Pseudomonas aeruginosa. Here, we present the structure and mechanism of P. aeruginosa SAH showing that the enzyme promiscuously hydrolyses (p)ppGpp and (p)ppApp in a strictly manganese‐dependent manner. While being dispensable for P. aeruginosa growth or swimming, swarming, and twitching motilities, its enzymatic activity is required for biofilm formation. Moreover, (p)ppApp‐degradation by SAH provides protection against the T6SS (p)ppApp synthetase effector Tas1, suggesting that SAH enzymes can also serve as defense proteins during interbacterial competition.
The genome of Pseudomonas aeruginosa encodes, besides the long RSH enzymes RelA and SpoT, the small alarmone hydrolase SAH that hydrolyses (p)ppGpp and the related (p)ppApp molecules in vitro in a manganese‐dependent manner. SAH is required for biofilm formation of P. aeruginosa and attenuates the toxicity of the type VI secretion system effector protein and (p)ppApp synthetase Tas1 during interbacterial competition. This study thus highlights the functional relevance of SAH enzymes for bacteria.
Nonribosomal peptide synthetases (NRPSs) employ multiple domains, specifically arranged in modules, for the assembly‐line biosynthesis of a plethora of bioactive peptides. It is poorly understood how ...catalysis is correlated with the domain interplay and associated conformational changes. We developed FRET sensors of an elongation module to study in solution the intramodular interactions of the peptidyl carrier protein (PCP) with adenylation (A) and condensation (C) domains. Backed by HDX‐MS analysis, we discovered dynamic mixtures of conformations that undergo distinct population changes in favor of the PCP‐A and PCP‐C interactions upon completion of the adenylation and thiolation reactions, respectively. To probe this model we blocked PCP binding to the C domain by photocaging and triggered peptide bond formation with light. Changing intramodular domain affinities of the PCP appear to result in conformational shifts according to the logic of the templated assembly process.
Translocation of the peptidyl carrier protein (PCP) between two catalytic domains in nonribosomal peptide synthetases (NRPSs) has been studied for the first time. Monitoring PCP interaction with A and C domains during catalysis using two FRET sensors of an elongation module revealed conformational population shifts that correlate with the directionality of synthesis. The first light‐controlled NRPS was created to further probe and support this model.
The stringent response enables bacteria to respond to nutrient limitation and other stress conditions through production of the nucleotide-based second messengers ppGpp and pppGpp, collectively known ...as (p)ppGpp. Here, we report that (p)ppGpp inhibits the signal recognition particle (SRP)-dependent protein targeting pathway, which is essential for membrane protein biogenesis and protein secretion. More specifically, (p)ppGpp binds to the SRP GTPases Ffh and FtsY, and inhibits the formation of the SRP receptor-targeting complex, which is central for the coordinated binding of the translating ribosome to the SecYEG translocon. Cryo-EM analysis of SRP bound to translating ribosomes suggests that (p)ppGpp may induce a distinct conformational stabilization of the NG domain of Ffh and FtsY in Bacillus subtilis but not in E. coli.
Cyclic di-GMP (c-di-GMP) is a second messenger that modulates multiple responses to environmental and cellular signals in bacteria. Here we identify CdbA, a DNA-binding protein of the ...ribbon-helix-helix family that binds c-di-GMP in Myxococcus xanthus. CdbA is essential for viability, and its depletion causes defects in chromosome organization and segregation leading to a block in cell division. The protein binds to the M. xanthus genome at multiple sites, with moderate sequence specificity; however, its depletion causes only modest changes in transcription. The interactions of CdbA with c-di-GMP and DNA appear to be mutually exclusive and residue substitutions in CdbA regions important for c-di-GMP binding abolish binding to both c-di-GMP and DNA, rendering these protein variants non-functional in vivo. We propose that CdbA acts as a nucleoid-associated protein that contributes to chromosome organization and is modulated by c-di-GMP, thus revealing a link between c-di-GMP signaling and chromosome biology.
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