Biology subjects

Luisi, B.

Publications and source records attributed to Luisi, B..

8 recordsLinked to original sources

bioRxiv2026

The multidrug transporter Pdr5 from Saccharomyces cerevisiae is an ergosterol-modulated efflux pump

The pleiotropic drug resistance ABC transporter Pdr5 and its homologs play a critical role in multidrug resistance of numerous fungi, including pathogens. Previous studies demonstrated that alterations in the composition of the plasma membrane in which Pdr5 is embedded, specifically the ergosterol content, affected transport activity. Here we report the cryo-EM map of Saccharomyces cerevisiae Pdr5 in complex with ATP/ADP at 2.2 [A] resolution, revealing densities on the surface of the transmembrane domains as well as inside the substrate-binding site, that were assigned as ergosterol, identified and quantified by GC-MS, and the sphingolipid M(IP)2C, identified via native MS. The ergosterol molecules are bound to the protein in well-defined grooves and unexpected locations. We found that the activity of Pdr5 follows a Gaussian-like distribution, increasing with the ergosterol-concentration to reach a maximum before declining at higher concentrations, but this modulation was not observed in the presence of cholesterol, the sterol analogue found in mammalian cells. Mutational analysis corroborates the role of a single ergosterol-binding site in the modulation of Pdr5 activity. Our structural analysis identifies a protein-lipid network that could serve as a center for lipid engagement linking the surrounding lipid environment to the substrate-binding region of Pdr5. These findings advance our understanding of Pdr5 function by demonstrating that the lipid environment is closely linked to Pdr5 structure and activity.

biochemistry↗

bioRxiv2024

Transcriptome-scale analysis uncovers conserved residues in the hydrophobic core of the bacterial RNA chaperone Hfq required for small regulatory RNA stability

The RNA-chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal molecular architecture of the Hfq hexamer contains three well characterised surfaces which allow it to bind sRNAs to stabilise them and engage target transcripts. Hfq-interacting sRNAs are categorised into two classes based on the surfaces they use to bind Hfq. By characterising a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen starvation, we corroborated the important role of the three RNA binding surfaces for Hfq function. Surprisingly, we uncovered two conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfqs RNA binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilisation of both sRNA classes. However, the alanine substitutions at these residues had no measurable impact on protein stability, structure or equilibrium binding to target sRNAs in vitro. We propose that V22 and G34 are key to the cooperative function among the RNA-binding surfaces of Hfq, a mechanism especially critical under cellular conditions when there is an increased demand for Hfq.

microbiology↗

bioRxiv2024

Plastid translocon recycling in dinoflagellates demonstrates the portability of complex plastids between hosts

The plastids of photosynthetic organisms on land are predominantly primary plastids derived from an ancient endosymbiosis of a cyanobacterium. Conversely, marine photosynthetic diversity is dominated by plastids gained by subsequent endosymbioses of photosynthetic eukaryotes, so-called complex plastids. The plastids of major eukaryotic lineages including cryptophytes, haptophytes, stramenopiles, dinoflagellates and apicomplexans, were originally all posited to derive from a single secondary endosymbiosis of a red alga--the chromalveloate hypothesis 1. Subsequent phylogenetic resolution of eukaryotes indicated that separate events of plastid acquisition must have occurred to account for this distribution of plastids 2,3. The number of such events, however, and the donor organisms for the new plastid endosymbioses are still not resolved. A perceived bottleneck of endosymbiotic plastid gain is the development of protein targeting from the hosts into new plastids, and this supposition has often driven hypotheses towards minimising the number of plastid-gain events to explain plastid distribution in eukaryotes. But how plastid protein-targeting is established for new endosymbionts is often unclear, which makes it difficult to assess the likelihood of plastid transfers between lineages. Here we show that Kareniaceae dinoflagellates, that possess complex plastids known to be derived from haptophytes, acquired all the necessary protein import machinery from these haptophytes. Furthermore, cryo-electron tomography revealed that no additional membranes were added to the Kareniaceae complex plastid during serial endosymbiosis, suggesting that the haptophyte-derived import processes were sufficient. Our analyses suggests that complex red plastids are preadapted for horizontal transmission, potentially explaining their widespread distribution in aquatic algal diversity.

evolutionary biology↗

bioRxiv2023

Structural basis for human mitochondrial tRNA maturation

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), mt-RNase P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for a tRNA methyltransferase complex in mt-RNase P cleavage activity is not fully understood. Moreover, the mechanisms of pre-tRNA 3-cleavage and CCA addition have also yet to be uncovered. Here we report cryo-EM structures that visualise these events, revealing the molecular basis for the first steps of RNA-transcript processing in human mitochondria. Altogether, the structures visualise four steps of mitochondrial tRNA maturation: 5 and 3 tRNA-end processing, methylation and 3-CCA addition, and explain the defined sequential order of tRNA processing. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA processing and 3-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-variable mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes.

biochemistry↗

bioRxiv2022

Polymorphic ribonucleoprotein folding as a basis for translational regulation

The widely occurring bacterial RNA chaperone Hfq is a key factor in the post-transcriptional control of hundreds of genes in Pseudomonas aeruginosa. How this broadly acting protein can contribute to the regulation requirements of so many different genes remains puzzling. Here, we describe the structures of higher-order assemblies formed on control regions of different P. aeruginosa target mRNAs by Hfq and its partner protein Crc. Our results show that these assemblies have mRNA-specific quaternary architectures resulting from the combination of multivalent protein-protein interfaces and recognition of patterns in the RNA sequence. The structural polymorphism of the ribonucleoprotein assemblies enables selective translational repression of many different target mRNAs. This system suggests how highly complex regulatory pathways can evolve and be rewired with a simple economy of proteinogenic components. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/480102v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1b34242org.highwire.dtl.DTLVardef@1c31071org.highwire.dtl.DTLVardef@16c8750org.highwire.dtl.DTLVardef@27ca3c_HPS_FORMAT_FIGEXP M_FIG C_FIG The RNA chaperone Hfq, in conjunction with the co-repressor Crc, forms higher order assemblies on nascent mRNAs. These complexes impact on translation of hundreds of transcripts in the pathogen Pseudomonas aeruginosa. Assemblies with different quaternary structures result from the interactions of the proteins with sequence motifs and structural elements in different mRNA targets, as well as from a repertoire of protein-to-protein interfaces. In this way, the combination of RNA sequence and two proteins can generate the diversity required to regulate many genes. It is proposed that the multi-step assembly process is highly cooperative and most likely competes kinetically with translation initiation to silence the targeted transcripts.

molecular biology↗

bioRxiv2021

Acetylation regulates the oligomerization state and activity of RNase J, the major ribonuclease of Helicobacter pylori

In Helicobacter pylori, post-transcriptional regulation strongly relies on the activity of an RNA degradosome, composed of the essential ribonuclease RNase J and the DEAD-box RNA helicase RhpA. Here, we describe post-translational modifications of this protein complex that affect its activity. Cell-extracted RNase J is acetylated on multiple residues, one of which, K649, strongly impacts RNase J oligomerization, which in turn influences ribonuclease activity. Corroborating the link between oligomerization and activity, mutations targeting K649 and other residues affect the dimerization and in vitro activity of RNase J. Our crystal structure of RNase J reveals three loops that gate access to the active site and rationalizes how oligomerization state influences activity. The acetylated residues of RNase J are important for H. pylori morphology, highlighting that the modifications affect the RNase J cellular function. We propose acetylation as a regulatory level controlling the activity of RNase J and the H. pylori RNA degradosome.

microbiology↗

bioRxiv2020

Antisense oligonucleotides target a nearly invariant structural element from the SARS-CoV-2 genome and drive RNA degradation

RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stemloop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3 UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 infection in cell culture. Our results thus permit us to suggest that the s2m element is a site of vulnerability readily targeted by ASOs, which show promise as anti-viral agents.

molecular biology↗

bioRxiv2020

In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution

In Gram-negative bacteria, tripartite efflux pump AcrAB-TolC plays a prominent role in antibiotic resistance. We have used high resolution cryo-ET to visualize the structure of Escherichia coli AcrAB-TolC at a 7 [A] resolution in intact cells. The resulting structures show the detailed architecture of the assembled complex embedded into cell envelope. Interactions with the inner membrane enable crosstalk between AcrB and TolC through AcrA, suggesting that assembly in the native cellular environment is critical for the pump activation mechanism, where the allosteric activating signal is triggered by the alternate binding of AcrA to the lipid membrane and AcrB porter domain. We establish a platform for high resolution in situ structural studies of bacteria efflux pump, which can yield critical information in understanding complex assemblies function. One sentence summaryA 7 [A] in situ structure of the AcrAB-TolC pump from bacteria by cryo-ET reveals mechanisms for active efflux.

biophysics↗