Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[25452]
Open Access
Abstract: Phages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.
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May 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
Krios II-Titan Krios II at Diamond
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Andre
Schutzer Godoy
,
Aline Minalli
Nakamura
,
Alice
Douangamath
,
Yun
Song
,
Gabriela
Dias Noske
,
Victor
Oliveira Gawriljuk
,
Rafaela
Sachetto Fernandes
,
Humberto
D'Muniz Pereira
,
Ketllyn irene
Zagato Oliveira
,
Daren
Fearon
,
Alexandre
Dias
,
Tobias
Krojer
,
Michael
Fairhead
,
Alisa
Powell
,
Louise
Dunnett
,
Jose
Brandao-Neto
,
Rachael
Skyner
,
Rod
Chalk
,
Dávid
Bajusz
,
Miklós
Bege
,
Anikó
Borbás
,
György Miklós
Keserű
,
Frank
Von Delft
,
Glaucius
Oliva
Diamond Proposal Number(s):
[27083, 27023]
Open Access
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.
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Apr 2023
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Krios II-Titan Krios II at Diamond
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Gabriela
Dias Noske
,
Yun
Song
,
Rafaela
Sachetto Fernandes
,
Rod
Chalk
,
Haitem
Elmassoudi
,
Lizbe
Koekemoer
,
C. David
Owen
,
Tarick J.
El-Baba
,
Carol V.
Robinson
,
Glaucius
Oliva
,
Andre
Schutzer Godoy
Diamond Proposal Number(s):
[27083, 29349]
Open Access
Abstract: The main protease from SARS-CoV-2 (Mpro) is responsible for cleavage of the viral polyprotein. Mpro self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S Mpro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of Mpro N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the Mpro dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.
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Mar 2023
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[23268]
Open Access
Abstract: Protein filaments are used in myriads of ways to organize other molecules within cells. Some filament-forming proteins couple the hydrolysis of nucleotides to their polymerization cycle, thus powering the movement of other molecules. These filaments are termed cytomotive. Only members of the actin and tubulin protein superfamilies are known to form cytomotive filaments. We examined the basis of cytomotivity via structural studies of the polymerization cycles of actin and tubulin homologs from across the tree of life. We analyzed published data and performed structural experiments designed to disentangle functional components of these complex filament systems. Our analysis demonstrates the existence of shared subunit polymerization switches among both cytomotive actins and tubulins, i.e., the conformation of subunits switches upon assembly into filaments. These cytomotive switches can explain filament robustness, by enabling the coupling of kinetic and structural polarities required for cytomotive behaviors and by ensuring that single cytomotive filaments do not fall apart.
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Mar 2023
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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 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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I04-Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[24948, 31384]
Open Access
Abstract: A key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor-binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent secondary infection by the same or a closely related phage and nonproductive phage adsorption to lysed cell fragments, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA–Llp structure reveals that Llp is periplasmic and binds to a nonnative conformation of the plug of FhuA, causing the inward folding of two extracellular loops via “reverse” allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of Escherichia coli by phage T5 and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.
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Oct 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 I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
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Yanan
Zhu
,
Christopher W.
Koo
,
C. Keith
Cassidy
,
Matthew C.
Spink
,
Tao
Ni
,
Laura C.
Zanetti-Domingues
,
Benji
Bateman
,
Marisa
Martin-Fernandez
,
Juan
Shen
,
Yuewen
Sheng
,
Yun
Song
,
Zhengyi
Yang
,
Amy C.
Rosenzweig
,
Peijun
Zhang
Diamond Proposal Number(s):
[21004, 29812]
Open Access
Abstract: Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.
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Sep 2022
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[21404, 27980]
Open Access
Abstract: Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants1,2,3. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space4,5,6,7,8,9. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline–proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development.
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Sep 2022
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