I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[25402]
Open Access
Abstract: In the opportunistic human pathogen Pseudomonas aeruginosa (Pae), carbon catabolite repression (CCR) orchestrates the hierarchical utilization of N and C sources, and impacts virulence, antibiotic resistance and biofilm development. During CCR, the RNA chaperone Hfq and the catabolite repression control protein Crc form assemblies on target mRNAs that impede translation of proteins involved in uptake and catabolism of less preferred C sources. After exhaustion of the preferred C-source, translational repression of target genes is relieved by the regulatory RNA CrcZ, which binds to and acts as a decoy for Hfq. Here, we asked whether Crc action can be modulated to relieve CCR after exhaustion of a preferred carbon source. As Crc does not bind to RNA per se, we endeavored to identify an interacting protein. In vivo co-purification studies, co-immunoprecipitation and biophysical assays revealed that Crc binds to Pae strain O1 protein PA1677. Our structural studies support bioinformatics analyzes showing that PA1677 belongs to the isochorismatase-like superfamily. Ectopic expression of PA1677 resulted in de-repression of Hfq/Crc controlled target genes, while in the absence of the protein, an extended lag phase is observed during diauxic growth on a preferred and a non-preferred carbon source. This observations indicate that PA1677 acts as an antagonist of Crc that favors synthesis of proteins required to metabolize non-preferred carbon sources. We present a working model wherein PA1677 diminishes the formation of productive Hfq/Crc repressive complexes on target mRNAs by titrating Crc. Accordingly, we propose the name CrcA (catabolite repression control protein antagonist) for PA1677.
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May 2023
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I03-Macromolecular Crystallography
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Open Access
Abstract: Microbes that have evolved to live on lignocellulosic biomass face unique challenges in the effective and efficient use of this material as food. The bacterium Shewanella sp. ANA-3 has the potential to utilize arabinan and arabinoxylan, and uptake of the monosaccharide, l-arabinose, derived from these polymers, is known to be mediated by a single ABC transporter. We demonstrate that the substrate binding protein of this system, GafASw, binds specifically to l-arabinofuranose, which is the rare furanose form of l-arabinose found in lignocellulosic biomass. The structure of GafASw was resolved to 1.7 Å and comparison to Escherichia coli YtfQ (GafAEc) revealed binding site adaptations that confer specificity for furanose over pyranose forms of monosaccharides, while selecting arabinose over another related monosaccharide, galactose. The discovery of a bacterium with a natural predilection for a sugar found abundantly in certain lignocellulosic materials suggests an intimate connection in the enzymatic release and uptake of the sugar, perhaps to prevent other microbes scavenging this nutrient before it mutarotates to l-arabinopyranose. This biological discovery also provides a clear route to engineer more efficient utilization of plant biomass components in industrial biotechnology.
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Mar 2023
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Abstract: Aims: Certain lactic acid bacteria (LAB) are known to have anti-inflammatory effects; however, hiochi bacteria, which are taxonomically classified as LAB and known to spoil a traditional Japanese alcoholic beverage, have not been studied in the same context. The aim of this study is to investigate the anti-inflammatory effects of hiochi bacteria strains and the underlying mechanisms. Methods and Results: We screened 45 strains of hiochi bacteria for anti-inflammatory effects and found that Lentilactobacillus hilgardii H-50 strongly inhibits lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in mouse splenocytes. This inhibition is attributed to its specific surface layer proteins (SLPs), which directly bind to LPS. Conclusions: The L. hilgardii H-50 strain exerts anti-inflammatory effects through its SLPs.
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Feb 2023
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[28394]
Open Access
Abstract: Stl, the master repressor of the Staphylococcus aureus pathogenicity islands (SaPIs), targets phage-encoded proteins to derepress and synchronize the SaPI and the helper phage life cycles. To activate their cycle, some SaPI Stls target both phage dimeric and phage trimeric dUTPases (Duts) as antirepressors, which are structurally unrelated proteins that perform identical functions for the phage. This intimate link between the SaPI’s repressor and the phage inducer has imposed an evolutionary optimization of Stl that allows the interaction with Duts from unrelated organisms. In this work, we structurally characterize this sophisticated mechanism of specialization by solving the structure of the prototypical SaPIbov1 Stl in complex with a prokaryotic and a eukaryotic trimeric Dut. The heterocomplexes with Mycobacterium tuberculosis and Homo sapiens Duts show the molecular strategy of Stl to target trimeric Duts from different kingdoms. Our structural results confirm the participation of the five catalytic motifs of trimeric Duts in Stl binding, including the C-terminal flexible motif V that increases the affinity by embracing Stl. In silico and in vitro analyses with a monomeric Dut support the capacity of Stl to recognize this third family of Duts, confirming this protein as a universal Dut inhibitor in the different kingdoms of life.
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Jan 2023
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake – the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, modelled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with Kd values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra- and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu-meso-DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli, suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp, which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes.
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Dec 2022
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Lorena
Zuliani-Alvarez
,
Morten L.
Govasli
,
Jane
Rasaiyaah
,
Chris
Monit
,
Stephen O.
Perry
,
Rebecca P.
Sumner
,
Simon
Mcalpine-Scott
,
Claire
Dickson
,
K. M.
Rifat Faysal
,
Laura
Hilditch
,
Richard J.
Miles
,
Frederic
Bibollet-Ruche
,
Beatrice H.
Hahn
,
Till
Boecking
,
Nikos
Pinotsis
,
Leo C.
James
,
David A.
Jacques
,
Greg J.
Towers
Diamond Proposal Number(s):
[8547, 11235]
Open Access
Abstract: Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.
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Nov 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Tim
Nierhaus
,
Stephen H.
Mclaughlin
,
Frank
Bürmann
,
Danguole
Kureisaite-Ciziene
,
Sarah L.
Maslen
,
J. Mark
Skehel
,
Conny W. H.
Yu
,
Stefan M. V.
Freund
,
Louise F. H.
Funke
,
Jason W.
Chin
,
Jan
Lowe
Diamond Proposal Number(s):
[15916, 21426]
Abstract: During bacterial cell division, filaments of tubulin-like FtsZ form the Z-ring, which is the cytoplasmic scaffold for divisome assembly. In Escherichia coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits divisome components, including bitopic FtsN. FtsN regulates the periplasmic peptidoglycan synthase FtsWI. To characterize how FtsA regulates FtsN, we applied electron microscopy to show that E. coli FtsA forms antiparallel double filaments on lipid monolayers when bound to the cytoplasmic tail of FtsN. Using X-ray crystallography, we demonstrate that Vibrio maritimus FtsA crystallizes as an equivalent double filament. We identified an FtsA–FtsN interaction site in the IA–IC interdomain cleft of FtsA using X-ray crystallography and confirmed that FtsA forms double filaments in vivo by site-specific cysteine cross-linking. FtsA–FtsN double filaments reconstituted in or on liposomes prefer negative Gaussian curvature, like those of MreB, the actin-like protein of the elongasome. We propose that curved antiparallel FtsA double filaments together with treadmilling FtsZ filaments organize septal peptidoglycan synthesis in the division plane.
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Sep 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1229]
Abstract: The enterohemorrhagic Escherichia coli pathotype is responsible for severe and dangerous infections in humans. Establishment of the infection requires colonization of the gastro-intestinal tract, which is dependent on the Type III Secretion System. The Type III Secretion System (T3SS) allows attachment of the pathogen to the mammalian host cell and cytoskeletal rearrangements within the host cell. Blocking the functionality of the T3SS is likely to reduce colonization and therefore limit the disease. This route offers an alternative to antibiotics, and problems with the development of antibiotics resistance. Salicylidene acylhydrazides have been shown to have an inhibitory effect on the T3SS in several pathogens. However, the main target of these compounds is still unclear. Past work has identified a number of putative protein targets of these compounds, one of which being WrbA. Whilst WrbA is considered an off-target interaction, this study presents the effect of the salicylidne acylhydrazide compounds on the activity of WrbA, along with crystal structures of WrbA from Yersinia pseudotuberculosis and Salmonella serovar Typhimurium; the latter also containing parts of the compound in the structure. We also present data showing that the original compounds were unstable in acidic conditions, and that later compounds showed improved stability.
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Jul 2022
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I04-Macromolecular Crystallography
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Stacey L.
Heaver
,
Henry H.
Le
,
Peijun
Tang
,
Arnaud
Basle
,
Claudia
Mirretta Barone
,
Dai Long
Vu
,
Jillian L.
Waters
,
Jon
Marles-Wright
,
Elizabeth L.
Johnson
,
Dominic J.
Campopiano
,
Ruth E.
Ley
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, using genomic and biochemical approaches, we investigated the gene cluster for inositol lipid synthesis in BT using a previously undescribed strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol dihydroceramide, determined the crystal structure of the recombinant BT MIP synthase enzyme and identified the phosphatase responsible for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP-DAG) to phosphatidylinositol (PI-DAG). In vitro, loss of inositol lipid production altered BT capsule expression and antimicrobial peptide resistance. In vivo, loss of inositol lipids decreased bacterial fitness in a gnotobiotic mouse model. We identified a second putative, previously undescribed pathway for bacterial PI-DAG synthesis without a PIP-DAG intermediate, common in Prevotella. Our results indicate that inositol sphingolipid production is widespread in host-associated Bacteroidetes and has implications for symbiosis.
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Jun 2022
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I04-Macromolecular Crystallography
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Open Access
Abstract: Gram-negative pathogens like Burkholderia pseudomallei use trimeric autotransporter adhesins such as BpaC as key molecules in their pathogenicity. Our 1.4 Å crystal structure of the membrane proximal part of the BpaC head domain shows that the domain is exclusively made of left-handed parallel β-roll repeats. This, the largest such structure solved, has two unique features. First, the core, rather than being composed of the canonical hydrophobic Ile and Val, is made up primarily of the hydrophilic Thr and Asn, with two different solvent channels. Second, comparing BpaC to all other left-handed parallel β-roll structures showed that the position of the head domain in the protein correlates with the number and type of charged residues. In BpaC, only negatively charged residues face the solvent – in stark contrast to the primarily positive surface charge of the left-handed parallel β-roll “type” protein, YadA. We propose extending the definitions of these head domains to include the BpaC-like head domain as a separate subtype, based on its unusual sequence, position and charge. We speculate that the function of left-handed parallel β-roll structures may differ depending on their position in the structure.
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Jun 2022
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