Supramolecular assembly of the Escherichia coli LdcI upon acid stress.

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.

Dual function of UPF3B in early and late translation termination.

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance pathway that recognizes and degrades mRNAs with premature termination codons (PTCs). The mechanisms underlying translation termination are key to the understanding of RNA surveillance mechanisms such as NMD and crucial for the development of therapeutic strategies for NMD-related diseases. Here, we have used a fully reconstituted in vitro translation system to probe the NMD proteins for interaction with the termination apparatus. We discovered that UPF3B (i) interacts with the release factors, (ii) delays translation termination and (iii) dissociates post-termination ribosomal complexes that are devoid of the nascent peptide. Furthermore, we identified UPF1 and ribosomes as new interaction partners of UPF3B. These previously unknown functions of UPF3B during the early and late phases of translation termination suggest that UPF3B is involved in the crosstalk between the NMD machinery and the PTC-bound ribosome, a central mechanistic step of RNA surveillance.

Structure of a human cap-dependent 48S translation pre-initiation complex.

Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition.

Ribosome-SRP-FtsY cotranslational targeting complex in the closed state.

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome-nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP-RNC complex results in GTP-independent binding of the SRP-FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP-FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP-FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.

A network of SMG-8, SMG-9 and SMG-1 C-terminal insertion domain regulates UPF1 substrate recruitment and phosphorylation.

Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD effector UPF1 by the phosphoinositide-3-kinase-like kinase (PIKK) SMG-1 is a key step in NMD and occurs when SMG-1, its two regulatory factors SMG-8 and SMG-9, and UPF1 form a complex at a terminating ribosome. Electron cryo-microscopy of the SMG-1-8-9-UPF1 complex shows the head and arm architecture characteristic of PIKKs and reveals different states of UPF1 docking. UPF1 is recruited to the SMG-1 kinase domain and C-terminal insertion domain, inducing an opening of the head domain that provides access to the active site. SMG-8 and SMG-9 interact with the SMG-1 C-insertion and promote high-affinity UPF1 binding to SMG-1-8-9, as well as decelerated SMG-1 kinase activity and enhanced stringency of phosphorylation site selection. The presence of UPF2 destabilizes the SMG-1-8-9-UPF1 complex leading to substrate release. Our results suggest an intricate molecular network of SMG-8, SMG-9 and the SMG-1 C-insertion domain that governs UPF1 substrate recruitment and phosphorylation by SMG-1 kinase, an event that is central to trigger mRNA decay.

Membrane protein insertion and proton-motive-force-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC.

The SecY/61 complex forms the protein-channel component of the ubiquitous protein secretion and membrane protein insertion apparatus. The bacterial version SecYEG interacts with the highly conserved YidC and SecDF-YajC subcomplex, which facilitates translocation into and across the membrane. Together, they form the holo-translocon (HTL), which we have successfully overexpressed and purified. In contrast to the homo-dimeric SecYEG, the HTL is a hetero-dimer composed of single copies of SecYEG and SecDF-YajC-YidC. The activities of the HTL differ from the archetypal SecYEG complex. It is more effective in cotranslational insertion of membrane proteins and the posttranslational secretion of a ß-barreled outer-membrane protein driven by SecA and ATP becomes much more dependent on the proton-motive force. The activity of the translocating copy of SecYEG may therefore be modulated by association with different accessory subcomplexes: SecYEG (forming SecYEG dimers) or SecDF-YajC-YidC (forming the HTL). This versatility may provide a means to refine the secretion and insertion capabilities according to the substrate. A similar modularity may also be exploited for the translocation or insertion of a wide range of substrates across and into the endoplasmic reticular and mitochondrial membranes of eukaryotes.

Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex.

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse "effectors" that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2. In ternary CBC-ARS2 complexes with PHAX, NCBP3, or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that in vivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.

4-Anilinoquinazoline Derivatives as the First Potent NOD1-RIPK2 Signaling Pathway Inhibitors at the Nanomolar Range.

Inflammation is a defense mechanism that restores tissue damage and eliminates pathogens. Among the pattern recognition receptors that recognize danger or pathogenic signals, nucleotide oligomerization domains 1 and 2 (NOD1/2) have been identified to play an important role in innate immunity responses, and inhibition of NOD1 could be interesting to treat severe infections and inflammatory diseases. In this work, we identified the first selective NOD1 versus NOD2 pathway inhibitors at the nanomolar range based on a 4-anilinoquinazoline scaffold. We demonstrated that NOD1 inhibition occurs through the inhibition of receptor interacting protein kinase 2 (RIPK2), which is involved in its downstream signaling pathways. Compound 37 demonstrates no cytotoxicity, a selectivity for RIPK2 over epithelial and vascular endothelial growth factor receptors (EGFR/VEGFR), and a capacity to reduce pro-inflammatory cytokine IL-8 secretion. The structure of the RIPK2-compound 37 complex was resolved by crystallography. The 4-anilinoquinazoline scaffold offers novel perspectives to design NOD1-RIPK2 signaling inhibitors.

Structure shows that the BIR2 domain of E3 ligase XIAP binds across the RIPK2 kinase dimer interface.

RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2-K209 residue in the RIPK2-XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination.

The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane.

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to aminoglycosides under anaerobiosis and engaged in cell membrane regulation.

Structural and biochemical characterisation of the Providencia stuartii arginine decarboxylase shows distinct polymerisation and regulation.

Bacterial homologous lysine and arginine decarboxylases play major roles in the acid stress response, physiology, antibiotic resistance and virulence. The Escherichia coli enzymes are considered as their archetypes. Whereas acid stress triggers polymerisation of the E. coli lysine decarboxylase LdcI, such behaviour has not been observed for the arginine decarboxylase Adc. Here we show that the Adc from a multidrug-resistant human pathogen Providencia stuartii massively polymerises into filaments whose cryo-EM structure reveals pronounced differences between Adc and LdcI assembly mechanisms. While the structural determinants of Adc polymerisation are conserved only in certain Providencia and Burkholderia species, acid stress-induced polymerisation of LdcI appears general for enterobacteria. Analysis of the expression, activity and oligomerisation of the P. stuartii Adc further highlights the distinct properties of this unusual protein and lays a platform for future investigation of the role of supramolecular assembly in the superfamily or arginine and lysine decarboxylases.

Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance.

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.

Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex.

The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that inside the LdcI-RavA cage, RavA hexamers adopt an asymmetric spiral conformation in which the nucleotide-free seam is constrained to two opposite orientations. Cryo-EM reconstructions of free RavA reveal two co-existing structural states: an asymmetric spiral, and a flat C2-symmetric closed ring characterised by two nucleotide-free seams. The closed ring RavA state bears close structural similarity to the pseudo two-fold symmetric crystal structure of the AAA+ unfoldase ClpX, suggesting a common ATPase mechanism. Based on these structures, and in light of the current knowledge regarding AAA+ ATPases, we propose different scenarios for the ATP hydrolysis cycle of free RavA and the LdcI-RavA cage-like complex, and extend the comparison to other AAA+ ATPases of clade 7.

Complete structure of the chemosensory array core signalling unit in an E. coli minicell strain.

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.

Src promotes delta opioid receptor (DOR) desensitization by interfering with receptor recycling.

Abstract An important limitation in the clinical use of opiates is progressive loss of analgesic efficacy over time. Development of analgesic tolerance is tightly linked to receptor desensitization. In the case of delta opioid receptors (DOR), desensitization is especially swift because receptors are rapidly internalized and are poorly recycled to the membrane. In the present study, we investigated whether Src activity contributed to this sorting pattern and to functional desensitization of DORs. A first series of experiments demonstrated that agonist binding activates Src and destabilizes a constitutive complex formed by the spontaneous association of DORs with the kinase. Src contribution to DOR desensitization was then established by showing that pre-treatment with Src inhibitor PP2 (20 microM; 1 hr) or transfection of a dominant negative Src mutant preserved DOR signalling following sustained exposure to an agonist. This protection was afforded without interfering with endocytosis, but suboptimal internalization interfered with PP2 ability to preserve DOR signalling, suggesting a post-endocytic site of action for the kinase. This assumption was confirmed by demonstrating that Src inhibition by PP2 or its silencing by siRNA increased membrane recovery of internalized DORs and was further corroborated by showing that inhibition of recycling by monensin or dominant negative Rab11 (Rab11S25N) abolished the ability of Src blockers to prevent desensitization. Finally, Src inhibitors accelerated recovery of DOR-Galphal3 coupling after desensitization. Taken together, these results indicate that Src dynamically regulates DOR recycling and by doing so contributes to desensitization of these receptors.

Apolipoproteins C-II and C-III inhibit selective uptake of low- and high-density lipoprotein cholesteryl esters in HepG2 cells.

Plasma low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors and are either totally degraded or their cholesteryl esters (CE) are selectively delivered to cells by receptors such as the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-II and apoC-III on the uptake of LDL and HDL by HepG2 cells. Stable transformants were obtained with sense or antisense strategies that secrete 47-294% the normal level of apoC-II or 60-200% that of apoC-III. Different levels of secreted apoC-II or apoC-III had little effect on LDL and HDL protein degradation by HepG2 cells. However, compared to controls, cells under-expressing apoC-II showed a 160% higher capacity to selectively take up HDL-CE, while cells under-expressing apoC-III demonstrated 70 and 160% higher capacity to take up CE from LDL and HDL, respectively. In experiments conducted with exogenously added apoC-II or apoC-III, no significant effect was observed on lipoprotein-protein association/degradation; however, LDL-CE and HDL-CE selective uptake was significantly reduced in a dose-dependent manner. These results indicate that apoC-II and apoC-III inhibit CE-selective uptake.

Structural basis of signal sequence surveillance and selection by the SRP-FtsY complex.

Signal-recognition particle (SRP)-dependent targeting of translating ribosomes to membranes is a multistep quality-control process. Ribosomes that are translating weakly hydrophobic signal sequences can be rejected from the targeting reaction even after they are bound to the SRP. Here we show that the early complex, formed by Escherichia coli SRP and its receptor FtsY with ribosomes translating the incorrect cargo EspP, is unstable and rearranges inefficiently into subsequent conformational states, such that FtsY dissociation is favored over successful targeting. The N-terminal extension of EspP is responsible for these defects in the early targeting complex. The cryo-electron microscopy structure of this 'false' early complex with EspP revealed an ordered M domain of SRP protein Ffh making two ribosomal contacts, and the NG domains of Ffh and FtsY forming a distorted, flexible heterodimer. Our results provide a structural basis for SRP-mediated signal-sequence selection during recruitment of the SRP receptor.

Localization and regulation of SR-BI in membrane rafts of HepG2 cells.

The scavenger receptor class B, type I (SR-BI) mediates cholesteryl esters (CE) selective uptake from low density lipoprotein (LDL) and high-density lipoprotein (HDL) particles. In a number of tissues expressing caveolin, SR-BI is localized in caveolae. We show using detergent-free sucrose gradients that SR-BI is found in membrane rafts devoid of caveolin-1 in the human hepatoma HepG2 cell. Perturbation of the structure of HepG2 cell membrane rafts with cholesterol oxidase or sphingomyelinase decreased LDL-CE association due to selective uptake by 60%, while HDL3-CE selective uptake was increased 2.3-fold by cholesterol oxidase but was not affected by sphingomyelinase. Sequestration of membrane cholesterol with filipin III decreased LDL-CE selective uptake by 25%, while it had no effect on HDL3-CE selective uptake. Extraction of cell membrane cholesterol with beta-cyclodextrin increased LDL- and HDL3-CE selective uptake by 1.6-fold and 3-fold, respectively. We found that CE-selective uptake from both HDL and LDL occurs by a pathway involving retro-endocytosis in HepG2 cells. An analysis of the effect of SR-BI level on the expression of critical lipid sensor and lipid binding proteins was conducted with stable transformants of HepG2 cell overexpressing SR-BI. We found that liver-type fatty acid binding protein expression level is higher in SR-BI-overexpressing cells and that caveolin-1 and sterol response element binding protein-2 levels are reduced. Thus, in this hepatic cell model, SR-BI is associated with membrane rafts devoid of caveolin and its expression affects intracellular lipid binding and lipid sensor proteins. SR-BI-dependent LDL- and HDL-CE selective uptake are affected differently by the integrity of membrane rafts, but both occur by a retroendocytic pathway in HepG2 cells.