I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[21970]
Open Access
Abstract: Oral and gut microbiomes are important for the maintenance of homeostasis in the human body. Altered or disturbed mutualism between their members results in dysbiosis with local injury and subsequent systemic diseases. The high bacterial density causes intense competition among microbiome residents to acquire nutrients, including iron and heme, the latter of high importance for heme auxotrophic members of the Bacteroidetes phylum. Our main hypothesis is that the heme acquisition mechanism, with the leading role played by a novel HmuY family of hemophore-like proteins, can be used to fulfill nutritional requirements and increase virulence. We characterized HmuY homologs expressed by Bacteroides fragilis and compared their properties with the first representative of this family, the HmuY protein of Porphyromonas gingivalis. In contrast to other Bacteroidetes members, B. fragilis produces three HmuY homologs (Bfr proteins). All bfr transcripts were produced at higher levels in bacteria starved of iron and heme (fold change increase ~60, ~90, and ~70 for bfrA, bfrB, and bfrC, respectively). X-ray protein crystallography showed that B. fragilis Bfr proteins are structurally similar to P. gingivalis HmuY and to other homologs, except for differences in the potential heme-binding pockets. BfrA binds heme, mesoheme, and deuteroheme, but preferentially under reducing conditions, using Met175 and Met146 to coordinate heme iron. BfrB binds iron-free protoporphyrin IX and coproporphyrin III, whereas BfrC does not bind porphyrins. HmuY is capable of heme sequestration from BfrA, which might increase the ability of P. gingivalis to cause dysbiosis also in the gut microbiome.
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Jul 2023
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[30015, 23316, 17844]
Open Access
Abstract: Sucrose import from photosynthetic tissues into the phloem is mediated by transporters from the low-affinity sucrose transporter family (SUC/SUT family). Furthermore, sucrose redistribution to other tissues is driven by phloem sap movement, the product of high turgor pressure created by this import activity. Additionally, sink organs such as fruits, cereals and seeds that accumulate high concentrations of sugar also depend on this active transport of sucrose. Here we present the structure of the sucrose–proton symporter, Arabidopsis thaliana SUC1, in an outward open conformation at 2.7 Å resolution, together with molecular dynamics simulations and biochemical characterization. We identify the key acidic residue required for proton-driven sucrose uptake and describe how protonation and sucrose binding are strongly coupled. Sucrose binding is a two-step process, with initial recognition mediated by the glucosyl moiety binding directly to the key acidic residue in a stringent pH-dependent manner. Our results explain how low-affinity sucrose transport is achieved in plants, and pinpoint a range of SUC binders that help define selectivity. Our data demonstrate a new mode for proton-driven symport with links to cation-driven symport and provide a broad model for general low-affinity transport in highly enriched substrate environments.
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May 2023
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B21-High Throughput SAXS
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[31323, 31668]
Open Access
Abstract: The allosteric regulation of biomolecules, such as enzymes, enables them to adapt and alter their conformation to fit specific substrates, expressing different functionalities in response to stimuli. Different stimuli can also trigger synthetic coordination cages to change their shape, size, and nuclearity by reconfiguring the dynamic metal–ligand bonds that hold them together. Here we demonstrate an abiological system consisting of different organic subcomponents and ZnII metal ions, which can respond to simple stimuli in complex ways. A ZnII20L12 dodecahedron transforms to give a larger ZnII30L12 icosidodecahedron through subcomponent exchange, as an aldehyde that forms bidentate ligands is displaced in favor of one that forms tridentate ligands together with a penta-amine subcomponent. In the presence of a chiral template guest, the same system that produced the icosidodecahedron instead gives a ZnII15L6 truncated rhombohedral architecture through enantioselective self-assembly. Under specific crystallization conditions, a guest induces a further reconfiguration of either the ZnII30L12 or ZnII15L6 cages to yield an unprecedented ZnII20L8 pseudo-truncated octahedral structure. The transformation network of these cages shows how large synthetic hosts can undergo structural adaptation through the application of chemical stimuli, opening pathways to broader applications.
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May 2023
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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
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Grace Q.
Gong
,
Benoit
Bilanges
,
Ben
Allsop
,
Glenn
Masson
,
Victoria
Roberton
,
Trevor
Askwith
,
Sally
Oxenford
,
Ralitsa R.
Madsen
,
Sarah E.
Conduit
,
Dom
Bellini
,
Martina
Fitzek
,
Matt
Collier
,
Osman
Najam
,
Zhenhe
He
,
Ben
Wahab
,
Stephen H.
Mclaughlin
,
A. W. Edith
Chan
,
Isabella
Feierberg
,
Andrew
Madin
,
Daniele
Morelli
,
Amandeep
Bhamra
,
Vanesa
Vinciauskaite
,
Karen E.
Anderson
,
Silvia
Surinova
,
Nikos
Pinotsis
,
Elena
Lopez-Guadamillas
,
Matthew
Wilcox
,
Alice
Hooper
,
Chandni
Patel
,
Maria A.
Whitehead
,
Tom D.
Bunney
,
Len R.
Stephens
,
Phillip T.
Hawkins
,
Matilda
Katan
,
Derek M.
Yellon
,
Sean M.
Davidson
,
David M.
Smith
,
James B.
Phillips
,
Richard
Angell
,
Roger L.
Williams
,
Bart
Vanhaesebroeck
Diamond Proposal Number(s):
[28677]
Abstract: Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
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May 2023
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19951]
Open Access
Abstract: Facultative anaerobic bacteria such as Escherichia coli have two α2β2 heterotetrameric trifunctional enzymes (TFE), catalyzing the last three steps of the β-oxidation cycle: soluble aerobic TFE (EcTFE) and membrane-associated anaerobic TFE (anEcTFE), closely related to the human mitochondrial TFE (HsTFE). The cryo-EM structure of anEcTFE and crystal structures of anEcTFE-α show that the overall assembly of anEcTFE and HsTFE is similar. However, their membrane-binding properties differ considerably. The shorter A5-H7 and H8 regions of anEcTFE-α result in weaker α-β as well as α-membrane interactions, respectively. The protruding H-H region of anEcTFE-β is therefore more critical for membrane-association. Mutational studies also show that this region is important for the stability of the anEcTFE-β dimer and anEcTFE heterotetramer. The fatty acyl tail binding tunnel of the anEcTFE-α hydratase domain, as in HsTFE-α, is wider than in EcTFE-α, accommodating longer fatty acyl tails, in good agreement with their respective substrate specificities.
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May 2023
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I24-Microfocus Macromolecular Crystallography
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Peter T.
Buckley
,
Rita
Chan
,
Jeffrey
Fernandez
,
Jinquan
Luo
,
Keenan A.
Lacey
,
Ashley L.
Dumont
,
Aidan
O’malley
,
Randall J.
Brezski
,
Songmao
Zheng
,
Thomas
Malia
,
Brian
Whitaker
,
Adam
Zwolak
,
Angela
Payne
,
Desmond
Clark
,
Martin
Sigg
,
Eilyn R.
Lacy
,
Anna
Kornilova
,
Debra
Kwok
,
Steve
Mccarthy
,
Bingyuan
Wu
,
Brian
Morrow
,
Jennifer
Nemeth-Seay
,
Ted
Petley
,
Sam
Wu
,
William R.
Strohl
,
Anthony S.
Lynch
,
Victor J.
Torres
Open Access
Abstract: Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein (“mAbtyrin”) that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.
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Apr 2023
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I24-Microfocus Macromolecular Crystallography
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Abstract: RAS proteins control various intracellular signaling networks. Mutations at specific locations were shown to stabilize their active guanosine triphosphate (GTP)-bound state, which is associated with the development of multiple cancers. An attractive approach to modulate RAS signaling is through its regulatory guanine nucleotide exchange factor (GEF) son of sevenless 1 (SOS1). With the recent discovery of Nanobody14 (Nb14), which potently enhances SOS1-catalyzed nucleotide exchange on RAS, we explored the feasibility of developing peptide mimetics by structurally mimicking the complementarity-determining region 3 (CDR3). Guided by a biochemical GEF assay and X-ray co-crystal structures, successive rounds of optimization and gradual conformational rigidification led to CDR3 mimetics showing half of the maximal activation potential of Nb14 with an EC50 value of 29 μM. Altogether, this study demonstrated that peptides able to modulate a protein-protein interaction can be obtained by structural mimicry of a Nb paratope.
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Apr 2023
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Evolution creates functional diversity of proteins, the essential building blocks of all biological systems. However, studies of natural proteins sampled across the tree of life and evaluated in a single experimental system are lacking. Almost half of enzymes require metals, and metalloproteins tend to optimally utilize the physicochemical properties of a specific metal co-factor. Life must adapt to changes in metal bioavailability, including those during the transition from anoxic to oxic Earth or pathogens’ exposure to nutritional immunity. These changes can challenge the ability of metalloenzymes to maintain activity, presumptively driving their evolution. Here we studied metal-preference evolution within the natural diversity of the iron/manganese superoxide dismutase (SodFM) family of reactive oxygen species scavengers. We identified and experimentally verified residues with conserved roles in determining metal preference that, when combined with an understanding of the protein’s evolutionary history, improved prediction of metal utilization across the five SodFM subfamilies defined herein. By combining phylogenetics, biochemistry and structural biology, we demonstrate that SodFM metal utilization can be evolutionarily fine tuned by sliding along a scale between perfect manganese and iron specificities. Over the history of life, SodFM metal preference has been modulated multiple independent times within different evolutionary and ecological contexts, and can be changed within short evolutionary timeframes.
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Apr 2023
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B21-High Throughput SAXS
I24-Microfocus Macromolecular Crystallography
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Kai
Wu
,
Tanya K.
Ronson
,
Pingru
Su
,
Zhi
Chen
,
Leonard
Goh
,
Andrew W.
Heard
,
Xiaopeng
Li
,
Fabian
Klautzsch
,
Christoph A.
Schalley
,
Mladen
Vinković
,
Jonathan R.
Nitschke
Diamond Proposal Number(s):
[31323, 31668]
Abstract: Biological encapsulants, such as viral capsids and ferritin protein cages, use many identical subunits to tile the surface of a polyhedron. Inspired by these natural systems, synthetic chemists have prepared artificial nanocages with well-defined shapes and cavities. Rational control over the self-assembly of discrete, nanometre-scale, hollow coordination cages composed of simple components remains challenging as a result of the entropic costs associated with binding many subunits together, difficulties in the error-correction processes associated with assembly and increasing surface energy as their size grows. Here we demonstrate the construction of nanocages of increasing size derived from a single pentatopic pyrrole-based subcomponent. Reasoned shifts in the preferred coordination number of the metal ions used, along with the denticity and steric hindrance of the ligands, enabled the generation of progressively larger cages. These structural changes of the cages are reminiscent of the differences in the folding of proteins caused by minor variations in their amino acid sequences; understanding how they affect capsule structure and thus cavity size may help to elucidate the construction principles for larger and functional capsules, capable of binding and carrying large biomolecules as cargoes.
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Apr 2023
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