Wieland Steinchen

The magic dance of the alarmones (p)ppGpp

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.

MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly.

Significance Flagella are bacterial organelles of locomotion. The number and location of flagella (flagellation pattern) are species specific and represent one of the earliest taxonomic criteria in microbiology. During each round of cell division, bacteria reproduce their flagellation pattern. FlhG is essential to a variety of flagellation patterns (e.g., polar, lateral) by yet-unknown mechanisms. We show that FlhG is an MinD-like ATPase that interacts with the flagellar C-ring proteins FliM/FliY in a nucleotide-independent manner. FlhG activates FliM/FliY to assemble with the C-ring protein FliG. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. We identify an underappreciated structural diversity of flagellar building blocks that contribute to formation of different flagellation patterns. The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.

Significance The alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named “(p)ppGpp)”] are important for the adaptation of bacteria and plant chloroplasts to a variety of environmental stress conditions. Their synthesis is carried out by (p)ppGpp synthetases. We delineate the catalytic mechanism of (p)ppGpp synthesis by oligomeric and highly cooperative small alarmone synthetase 1 (SAS1) at atomic resolution. Our structural and biochemical analysis shows that only pppGpp—but not ppGpp—positively affects the activity of SAS1. To our knowledge, this is the first molecular description of a biological activity in which pppGpp and ppGpp execute different functional roles. Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named “(p)ppGpp”] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp—but not ppGpp—positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

Structure of the Bacillus subtilis hibernating 100S ribosome reveals the basis for 70S dimerization

Under stress conditions, such as nutrient deprivation, bacteria enter into a hibernation stage, which is characterized by the appearance of 100S ribosomal particles. In Escherichia coli, dimerization of 70S ribosomes into 100S requires the action of the ribosome modulation factor (RMF) and the hibernation‐promoting factor (HPF). Most other bacteria lack RMF and instead contain a long form HPF (LHPF), which is necessary and sufficient for 100S formation. While some structural information exists as to how RMF and HPF mediate formation of E. coli 100S (Ec100S), structural insight into 100S formation by LHPF has so far been lacking. Here we present a cryo‐EM structure of the Bacillus subtilis hibernating 100S (Bs100S), revealing that the C‐terminal domain (CTD) of the LHPF occupies a site on the 30S platform distinct from RMF. Moreover, unlike RMF, the BsHPF‐CTD is directly involved in forming the dimer interface, thereby illustrating the divergent mechanisms by which 100S formation is mediated in the majority of bacteria that contain LHPF, compared to some γ‐proteobacteria, such as E. coli.

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

Significance Cyclic dimeric GMP (c-di-GMP) has emerged as ubiquitous bacterial second messenger, regulating multiple cellular functions, such as cell cycle, virulence, and biofilm formation. However, our knowledge on the molecular inventory, diversity, and function of c-di-GMP receptors, and the molecular evolution of c-di-GMP–responsive proteins is still incomplete. We have identified a class of c-di-GMP–responsive transcription factors, strikingly illustrating how a classical transcription factor has acquired the ability to sense this signaling molecule. The mode of c-di-GMP binding to the AraC-like transcription factor CuxR is highly reminiscent to that of the PilZ domain, the prototypic c-di-GMP receptor. PilZ and CuxR provide an example of convergent evolution in which c-di-GMP binding sites of similar topology have evolved independently in two distinct protein families. Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal “RxxxR” motif and a β-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP–responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP–responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins.

Computational method allowing Hydrogen-Deuterium Exchange Mass Spectrometry at single amide Resolution

Hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (HDXMS) is a rapid and effective method for localizing and determining protein stability and dynamics. Localization is routinely limited to a peptide resolution of 5 to 20 amino acid residues. HDXMS data can contain information beyond that needed for defining protein stability at single amide resolution. Here we present a method for extracting this information from an HDX dataset to generate a HDXMS protein stability fingerprint. High resolution (HR)-HDXMS was applied to the analysis of a model protein of a spectrin tandem repeat that exemplified an intuitive stability profile based on the linkage of two triple helical repeats connected by a helical linker. The fingerprint recapitulated expected stability maximums and minimums with interesting structural features that corroborate proposed mechanisms of spectrin flexibility and elasticity. HR-HDXMS provides the unprecedented ability to accurately assess protein stability at the resolution of a single amino acid. The determination of HDX stability fingerprints may be broadly applicable in many applications for understanding protein structure and function as well as protein ligand interactions.

FliS/flagellin/FliW heterotrimer couples type III secretion and flagellin homeostasis

Flagellin is amongst the most abundant proteins in flagellated bacterial species and constitutes the major building block of the flagellar filament. The proteins FliW and FliS serve in the post-transcriptional control of flagellin and guide the protein to the flagellar type III secretion system (fT3SS), respectively. Here, we present the high-resolution structure of FliS/flagellin heterodimer and show that FliS and FliW bind to opposing interfaces located at the N- and C-termini of flagellin. The FliS/flagellin/FliW heterotrimer is able to interact with FlhA-C suggesting that FliW and FliS are released during flagellin export. After release, FliW and FliS are recycled to execute a new round of post-transcriptional regulation and targeting. Taken together, our study provides a mechanism explaining how FliW and FliS synchronize the production of flagellin with the capacity of the fT3SS to secrete flagellin.

Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus

The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.

HDX-MS in den Lebenswissenschaften

In-depth structural and mechanistic analysis of biomolecular complexes is crucial for modern life sciences. Here we give an overview on hydrogendeuterium exchange mass spectrometry (HDX-MS) as an attractive tool to study the dynamic and structural features of proteins and their complexes.

Erratum zu: HDX-MS in den Lebenswissenschaften

Bei der Produktion der bereits veröffentlichten Originalversion des Artikels ist ein Fehler in der Legende zu Abbildung 1 entstanden. Die Farbbezeichnungen bei Wasserstoff (blau) und Deuterium (rot) waren vertauscht. Anbei finden Sie die korrigierte Version.Wir entschuldigen uns für den Fehler und für alle dadurch eventuell entstandenen Unannehmlichkeiten.Die Online-Version des Originalartikels finden Sie unter der DOI: 10.1007/s12268-017-0871-8.