Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[25127]
Open Access
Abstract: CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.
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Feb 2023
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[22238, 17057]
Abstract: The molecular mode of action of biguanides, including the drug metformin, which is widely used in the treatment of diabetes, is incompletely characterized. Here, we define the inhibitory drug-target interaction(s) of a model biguanide with mammalian respiratory complex I by combining cryo–electron microscopy and enzyme kinetics. We interpret these data to explain the selectivity of biguanide binding to different enzyme states. The primary inhibitory site is in an amphipathic region of the quinone-binding channel, and an additional binding site is in a pocket on the intermembrane-space side of the enzyme. An independent local chaotropic interaction, not previously described for any drug, displaces a portion of a key helix in the membrane domain. Our data provide a structural basis for biguanide action and enable the rational design of medicinal biguanides.
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Jan 2023
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
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Nisha
Pillay
,
Laura
Mariotti
,
Mariola
Zaleska
,
Oviya
Inian
,
Matthew
Jessop
,
Sam
Hibbs
,
Ambroise
Desfosses
,
Paul C. R.
Hopkins
,
Catherine M.
Templeton
,
Fabienne
Beuron
,
Edward P.
Morris
,
Sebastian
Guettler
Diamond Proposal Number(s):
[28549, 15624, 16822, 16023, 21809]
Open Access
Abstract: The poly-ADP-ribosyltransferase tankyrase (TNKS, TNKS2) controls a wide range of disease-relevant cellular processes, including WNT–β-catenin signalling, telomere length maintenance, Hippo signalling, DNA damage repair and glucose homeostasis1,2. This has incentivized the development of tankyrase inhibitors. Notwithstanding, our knowledge of the mechanisms that control tankyrase activity has remained limited. Both catalytic and non-catalytic functions of tankyrase depend on its filamentous polymerization3,4,5. Here we report the cryo-electron microscopy reconstruction of a filament formed by a minimal active unit of tankyrase, comprising the polymerizing sterile alpha motif (SAM) domain and its adjacent catalytic domain. The SAM domain forms a novel antiparallel double helix, positioning the protruding catalytic domains for recurring head-to-head and tail-to-tail interactions. The head interactions are highly conserved among tankyrases and induce an allosteric switch in the active site within the catalytic domain to promote catalysis. Although the tail interactions have a limited effect on catalysis, they are essential to tankyrase function in WNT–β-catenin signalling. This work reveals a novel SAM domain polymerization mode, illustrates how supramolecular assembly controls catalytic and non-catalytic functions, provides important structural insights into the regulation of a non-DNA-dependent poly-ADP-ribosyltransferase and will guide future efforts to modulate tankyrase and decipher its contribution to disease mechanisms.
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Nov 2022
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Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[15997, 22006]
Open Access
Abstract: Pyruvate carboxylase (PC) is a tetrameric enzyme that contains two active sites per subunit that catalyze two consecutive reactions. A mobile domain with an attached prosthetic biotin links both reactions, an initial biotin carboxylation and the subsequent carboxyl transfer to pyruvate substrate to produce oxaloacetate. Reaction sites are at long distance, and there are several co-factors that play as allosteric regulators. Here, using cryoEM we explore the structure of active PC tetramers focusing on active sites and on the conformational space of the oligomers. The results capture the mobile domain at both active sites and expose catalytic steps of both reactions at high resolution, allowing the identification of substrates and products. The analysis of catalytically active PC tetramers reveals the role of certain motions during enzyme functioning, and the structural changes in the presence of additional cofactors expose the mechanism for allosteric regulation.
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Oct 2022
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[21004, 20223, 21005]
Open Access
Abstract: Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. They encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase catalyzing carbon fixation inside a proteinaceous shell. How Rubisco complexes pack within the carboxysomes is unknown. Using cryo-electron tomography, we determine the distinct 3D organization of Rubisco inside two distant α-carboxysomes from a marine α-cyanobacterium Cyanobium sp. PCC 7001 where Rubiscos are organized in three concentric layers, and from a chemoautotrophic bacterium Halothiobacillus neapolitanus where they form intertwining spirals. We further resolve the structures of native Rubisco as well as its higher-order assembly at near-atomic resolutions by subtomogram averaging. The structures surprisingly reveal that the authentic intrinsically disordered linker protein CsoS2 interacts with Rubiscos in native carboxysomes but functions distinctively in the two α-carboxysomes. In contrast to the uniform Rubisco-CsoS2 association in the Cyanobium α-carboxysome, CsoS2 binds only to the Rubiscos close to the shell in the Halo α-carboxysome. Our findings provide critical knowledge of the assembly principles of α-carboxysomes, which may aid in the rational design and repurposing of carboxysome structures for new functions.
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Jul 2022
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[24039]
Open Access
Abstract: Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I-converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X-ray crystallography and molecular dynamics simulations. Here, we report the first cryo-EM structures of full-length, glycosylated, soluble sACE (sACES1211). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N- and C-terminal domains of monomeric sACES1211 were resolved at 3.7 and 4.1 Å, respectively, while the interacting N-terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators.
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Jul 2022
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Krios III-Titan Krios III at Diamond
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Manuel
Schweighauser
,
Diana
Arseni
,
Mehtap
Bacioglu
,
Melissa
Huang
,
Sofia
Lovestam
,
Yang
Shi
,
Yang
Yang
,
Wenjuan
Zhang
,
Abhay
Kotecha
,
Holly J.
Garringer
,
Ruben
Vidal
,
Grace I.
Hallin
,
Kathy L.
Newell
,
Airi
Tarutani
,
Shigeo
Murayama
,
Masayuki
Miyazaki
,
Yuko
Saito
,
Mari
Yoshida
,
Kazuko
Hasegawa
,
Tammaryn
Lashley
,
Tamas
Revesz
,
Gabor G.
Kovacs
,
John
Van Swieten
,
Masaki
Takao
,
Masato
Hasegawa
,
Bernardino
Ghetti
,
Maria Grazia
Spillantini
,
Benjamin
Ryskeldi-Falcon
,
Alexey G.
Murzin
,
Michel
Goedert
,
Sjors H. W.
Scheres
Diamond Proposal Number(s):
[17434, 23268]
Abstract: Many age-dependent neurodegenerative diseases, like Alzheimer’s and Parkinson’s, are characterised by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-β (Aβ), α-synuclein and TDP-43 are the most common1,2. Here, we used electron cryo-microscopy (cryo-EM) structure determination to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in human brains. We determined the cryo-EM structures of TMEM106B filaments from a number of brain regions of 22 individuals with abundant amyloid deposits, including sporadic and inherited tauopathies, Aβ-amyloidoses, synucleinopathies and TDP-43 proteinopathies, as well as from the frontal cortex of 3 neurologically normal individuals with no or only few amyloid deposits. We observed three TMEM106B folds, with no clear relationships between folds and diseases. TMEM106B filaments correlated with the presence of a 29 kDa sarkosyl-insoluble fragment and globular cytoplasmic inclusions, as detected by an antibody specific for the C-terminal region of TMEM106B. The identification of TMEM106B filaments in the brains of older, but not younger, neurologically normal individuals indicates that they form in an age-dependent manner.
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Mar 2022
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[23268]
Open Access
Abstract: AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor’s ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop (‘β1’), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.
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Feb 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 III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[18659]
Open Access
Abstract: The global spread of multidrug-resistant Acinetobacter baumannii infections urgently calls for the identification of novel drug targets. We solved the electron cryo-microscopy structure of the F1Fo–adenosine 5′-triphosphate (ATP) synthase from A. baumannii in three distinct conformational states. The nucleotide-converting F1 subcomplex reveals a specific self-inhibition mechanism, which supports a unidirectional ratchet mechanism to avoid wasteful ATP consumption. In the membrane-embedded Fo complex, the structure shows unique structural adaptations along both the entry and exit pathways of the proton-conducting a-subunit. These features, absent in mitochondrial ATP synthases, represent attractive targets for the development of next-generation therapeutics that can act directly at the culmination of bioenergetics in this clinically relevant pathogen.
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Feb 2022
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