I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Human topoisomerase II beta (TOP2B) modulates DNA topology using energy from ATP hydrolysis. To investigate the conformational changes that occur during ATP hydrolysis, we determined the X-ray crystallographic structures of the human TOP2B ATPase domain bound to AMPPNP or ADP at 1.9 Å and 2.6 Å resolution, respectively. The GHKL domains of both structures are similar, whereas the QTK loop within the transducer domain can move for product release. As TOP2B is the clinical target of bisdioxopiperazines, we also determined the structure of a TOP2B:ADP:ICRF193 complex to 2.3 Å resolution and identified key drug-binding residues. Biochemical characterization revealed the N-terminal strap reduces the rate of ATP hydrolysis. Mutagenesis demonstrated residue E103 as essential for ATP hydrolysis in TOP2B. Our data provide fundamental insights into the tertiary structure of the human TOP2B ATPase domain and a potential regulatory mechanism for ATP hydrolysis.
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Jun 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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ana S.
Luis
,
Arnaud
Basle
,
Dominic P.
Byrne
,
Gareth S. A.
Wright
,
James A.
London
,
Chunsheng
Jin
,
Niclas G.
Karlsson
,
Gunnar C.
Hansson
,
Patrick A.
Eyers
,
Mirjam
Czjzek
,
Tristan
Barbeyron
,
Edwin A.
Yates
,
Eric C.
Martens
,
Alan
Cartmell
Diamond Proposal Number(s):
[21970, 18598]
Abstract: Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.
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Jun 2022
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I04-Macromolecular Crystallography
I23-Long wavelength MX
I24-Microfocus Macromolecular Crystallography
Krios III-Titan Krios III at Diamond
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Paola
Lanzoni-Mangutchi
,
Oishik
Banerji
,
Jason
Wilson
,
Anna
Barwinska-Sendra
,
Joseph A.
Kirk
,
Filipa
Vaz
,
Shauna
O’beirne
,
Arnaud
Basle
,
Kamel
El Omari
,
Armin
Wagner
,
Neil F.
Fairweather
,
Gillian R.
Douce
,
Per A.
Bullough
,
Robert P.
Fagan
,
Paula
Salgado
Diamond Proposal Number(s):
[15523, 18598, 19832]
Open Access
Abstract: Many bacteria and archaea possess a two-dimensional protein array, or S-layer, that covers the cell surface and plays crucial roles in cell physiology. Here, we report the crystal structure of SlpA, the main S-layer protein of the bacterial pathogen Clostridioides difficile, and use electron microscopy to study S-layer organisation and assembly. The SlpA crystal lattice mimics S-layer assembly in the cell, through tiling of triangular prisms above the cell wall, interlocked by distinct ridges facing the environment. Strikingly, the array is very compact, with pores of only ~10 Å in diameter, compared to other S-layers (30–100 Å). The surface-exposed flexible ridges are partially dispensable for overall structure and assembly, although a mutant lacking this region becomes susceptible to lysozyme, an important molecule in host defence. Thus, our work gives insights into S-layer organisation and provides a basis for development of C. difficile-specific therapeutics.
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Feb 2022
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Krios I-Titan Krios I at Diamond
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Jennifer
Ross
,
Zak
Mciver
,
Thomas
Lambert
,
Cecilia
Piergentili
,
Jasmine Emma
Bird
,
Kelly J.
Gallagher
,
Faye L.
Cruickshank
,
Patrick
James
,
Efrain
Zarazúa-Arvizu
,
Louise E.
Horsfall
,
Kevin J.
Waldron
,
Marcus D.
Wilson
,
C. Logan
Mackay
,
Arnaud
Basle
,
David J.
Clarke
,
Jon
Marles-Wright
Diamond Proposal Number(s):
[16637]
Open Access
Abstract: Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo–electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.
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Jan 2022
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948, 13587, 18598]
Open Access
Abstract: Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed “methionine track” leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.
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Nov 2021
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure–function studies of metalloid porins, including the basis of their substrate selectivity.
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Oct 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ana S.
Luis
,
Chunsheng
Jin
,
Gabriel
Vasconcelos Pereira
,
Robert W. P.
Glowacki
,
Sadie R.
Gugel
,
Shaleni
Singh
,
Dominic P.
Byrne
,
Nicholas A.
Pudlo
,
James A.
London
,
Arnaud
Basle
,
Mark
Reihill
,
Stefan
Oscarson
,
Patrick A.
Eyers
,
Mirjam
Czjzek
,
Gurvan
Michel
,
Tristan
Barbeyron
,
Edwin A.
Yates
,
Gunnar C.
Hansson
,
Niclas G.
Karlsson
,
Alan
Cartmell
,
Eric C.
Martens
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease.
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Oct 2021
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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Marco
Salamina
,
Bailey C.
Montefiore
,
Mengxi
Liu
,
Daniel J.
Wood
,
Richard
Heath
,
James R.
Ault
,
Lan-Zhen
Wang
,
Svitlana
Korolchuk
,
Arnaud
Basle
,
Martyna
Pastok
,
Judith
Reeks
,
Natalie J.
Tatum
,
Frank
Sobott
,
Stefan T.
Arold
,
Michele
Pagano
,
Martin E. M.
Noble
,
Jane A.
Endicott
Diamond Proposal Number(s):
[13587, 16970]
Open Access
Abstract: The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.
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Mar 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Declan A.
Gray
,
Joshua B. R.
White
,
Abraham O.
Oluwole
,
Parthasarathi
Rath
,
Amy J.
Glenwright
,
Adam
Mazur
,
Michael
Zahn
,
Arnaud
Basle
,
Carl
Morland
,
Sasha L.
Evans
,
Alan
Cartmell
,
Carol V.
Robinson
,
Sebastian
Hiller
,
Neil A.
Ranson
,
David N.
Bolam
,
Bert
Van Den Berg
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a “pedal bin” transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.
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Jan 2021
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