Krios I-Titan Krios I at Diamond
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Jianbing
Ma
,
Gangshun
Yi
,
Mingda
Ye
,
Craig
Macgregor-Chatwin
,
Yuewen
Sheng
,
Ying
Lu
,
Ming
Li
,
Qingrong
Li
,
Dong
Wang
,
Robert J. C.
Gilbert
,
Peijun
Zhang
Diamond Proposal Number(s):
[29812]
Open Access
Abstract: The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous biomolecules and complexes. Nevertheless, cryoEM sample preparation of challenging proteins and complexes, especially those with low abundance or with preferential orientation, remains a major hurdle. We developed an affinity-grid method employing monodispersed single particle streptavidin on a lipid monolayer to enhance particle absorption on the grid surface and alleviate sample exposure to the air-water interface. Using this approach, we successfully enriched the Thermococcus kodakarensis mini-chromosome maintenance complex 3 (MCM3) on cryoEM grids through biotinylation and resolved its structure. We further utilized this affinity method to tether the biotin-tagged dsDNA to selectively enrich a stable MCM3-ATP-dsDNA complex for cryoEM structure determination. Intriguingly, both MCM3 apo and dsDNA bound structures exhibit left-handed open spiral conformations, distinct from other reported MCM structures. The large open gate is sufficient to accommodate a dsDNA which could potentially be melted. The value of mspSA affinity method was further demonstrated by mitigating the issue of preferential angular distribution of HIV-1 capsid protein hexamer and RNA polymerase II elongation complex from Saccharomyces cerevisiae.
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Dec 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[20223, 21004]
Open Access
Abstract: Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3′ end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A. Our structures reveal that pre-miRNAs engage the enzymes in fundamentally different ways depending on the presence of LIN28A, which clamps them onto the TUTs to enable processive 3′ oligo-uridylation. This study reveals the molecular basis for mono- versus oligo-uridylation by TUT7/4, as determined by the presence of LIN28A, and thus their mechanism of action in the regulation of cell fate and in cancer.
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Jul 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[20223, 21004]
Open Access
Abstract: Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre-assembled complete pre-pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre-pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β-hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre-pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.
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Oct 2022
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Perforin-like proteins (PLPs) play key roles in mechanisms associated with parasitic disease caused by apicomplexans parasites Plasmodium and Toxoplasma. The T. gondii PLP1 (TgPLP1) mediates tachyzoite egress from cells, while the five Plasmodium PLPs carry out various roles in the life cycle of the parasite and with respect to the molecular basis of disease. Here we focus on Plasmodium vivax PLP1 and PLP2 (PvPLP1 and PvPLP2) compared to TgPLP1. Determination of the crystal structure of the membrane-binding APCβ domain of PvPLP1 reveals notable differences with TgPLP1, reflected in its inability to bind lipid bilayers as TgPLP1 and PvPLP2 do. Molecular dynamics simulations combined with site-directed mutagenesis and functional assays allow dissection of the binding interactions of TgPLP1 and PvPLP2 on lipid bilayers, and reveal similar tropisms for lipids enriched in the inner leaflet of the mammalian plasma membrane. In addition PvPLP2 displays a secondary synergistic interaction side-on from its principal bilayer interface. This study underlines the substantial differences between the biophysical properties of the APCβ domains of apicomplexan PLPs, which reflect their significant sequence diversity. Such differences will be important factors in determining the cell targeting and membrane-binding activity of the different proteins in parasitic life cycles and disease.
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May 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Samuel C.
Griffiths
,
Rebekka A.
Schwab
,
Kamel
El Omari
,
Benjamin
Bishop
,
Ellen J.
Iverson
,
Tomas
Malinauskas
,
Ramin
Dubey
,
Mingxing
Qian
,
Douglas F.
Covey
,
Robert J. C.
Gilbert
,
Rajat
Rohatgi
,
Christian
Siebold
Diamond Proposal Number(s):
[19946, 14744]
Open Access
Abstract: Hedgehog (HH) morphogen signalling, crucial for cell growth and tissue patterning in animals, is initiated by the binding of dually lipidated HH ligands to cell surface receptors. Hedgehog-Interacting Protein (HHIP), the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling, binds directly to SHH with high nanomolar affinity, sequestering SHH. Here, we report the structure of the HHIP N-terminal domain (HHIP-N) in complex with a glycosaminoglycan (GAG). HHIP-N displays a unique bipartite fold with a GAG-binding domain alongside a Cysteine Rich Domain (CRD). We show that HHIP-N is required to convey full HHIP inhibitory function, likely by interacting with the cholesterol moiety covalently linked to HH ligands, thereby preventing this SHH-attached cholesterol from binding to the HH receptor Patched (PTCH1). We also present the structure of the HHIP C-terminal domain in complex with the GAG heparin. Heparin can bind to both HHIP-N and HHIP-C, thereby inducing clustering at the cell surface and generating a high-avidity platform for SHH sequestration and inhibition. Our data suggest a multimodal mechanism, in which HHIP can bind two specific sites on the SHH morphogen, alongside multiple GAG interactions, to inhibit SHH signalling.
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Dec 2021
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Tao
Ni
,
Fang
Jiao
,
Xiulian
Yu
,
Saša
Aden
,
Lucy
Ginger
,
Sophie I.
Williams
,
Fangfang
Bai
,
Vojtech
Prazak
,
Dimple
Karia
,
Phillip
Stansfeld
,
Peijun
Zhang
,
George
Munson
,
Gregor
Anderluh
,
Simon
Scheuring
,
Robert J. C.
Gilbert
Open Access
Abstract: Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.
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Jan 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Toxoplasma and Plasmodium are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The Toxoplasma gondii PLP1 (TgPLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of TgPLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by TgPLP1.
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Mar 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Hantaviruses are zoonotic pathogens with a near-global distribution that can cause severe hemorrhagic fever and pulmonary syndrome. The outer membrane of the hantavirus envelope displays a lattice of two glycoproteins, Gn and Gc, which orchestrate host cell recognition and entry. Here, we describe the crystal structure of the Gn glycoprotein ectodomain from the Asiatic Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Structural overlay analysis reveals that the HTNV Gn fold is highly similar to the Gn of Puumala virus (PUUV), a genetically and geographically distinct and less pathogenic hantavirus found predominantly in North-Eastern Europe, confirming that the hantaviral Gn fold is architecturally conserved across hantavirus clades. Interestingly, HTNV Gn crystallized at acidic pH, in a compact tetrameric configuration distinct from the organization at neutral pH. Analysis of the Gn, both in solution and in the context of the virion, confirms the pH-sensitive oligomeric nature of the glycoprotein, indicating that the hantaviral Gn undergoes structural transitions during host cell entry. These data allow us to present a structural model for how acidification during endocytic uptake of the virus triggers the dissociation of the metastable Gn-Gc lattice to enable insertion of the Gc-resident hydrophobic fusion loops into the host cell membrane. Together, these data reveal the dynamic plasticity of the structurally conserved hantaviral surface.
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Aug 2017
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain comprising F0-F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at 2.23Å resolution and characterise its lipid binding using biophysical and computational approaches. Molecular dynamics (MD) simulations suggest flexibility in the PH domain loops connecting β-strands forming the putative phosphatidylinositol phosphate (PtdInsP) binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the β1-β2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P3 compared to PtdIns(4,5)P2. Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as analyte indicate affinities of 300 μM for PtdIns(3,4,5)P3 and 1mM for PtdIns(4,5)P2. In contrast, SPR studies with immobilised PH domain and lipid nanodiscs as analyte show affinities of 0.40 µM for PtdIns(3,4,5)P3 and no affinity for PtdIns(4,5)P2 when the inositol phosphate constitutes 5% of the total lipids (~5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3 composition to 1% abolishes nanodisc binding to the PH domain, as does site-directed mutagenesis of two lysines within the β1-β2 loop. Binding of PtdIns(3,4,5)P3 by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3 clustering in the binding of some PH domains and not others, highlighting the importance lipid mobility and clustering for the biophysical assessment of protein-membrane interactions.
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Dec 2016
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Youxin
Kong
,
Bert J. C.
Janssen
,
Tomas
Malinauskas
,
Vamshidhar r.
Vangoor
,
Charlotte
Coles
,
Rainer
Kaufmann
,
Tao
Ni
,
Robert J. C.
Gilbert
,
Sergi
Padilla-Parra
,
R. jeroen
Pasterkamp
,
E. yvonne
Jones
Diamond Proposal Number(s):
[8423, 10627]
Open Access
Abstract: Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1–9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular “head-to-stalk” (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors.
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Aug 2016
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