I04-1-Macromolecular Crystallography (fixed wavelength)
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William M.
Browne
,
Jonathan
Pettinger
,
Teresa
Weckwerth
,
Andrew
Purkiss
,
Sing Hei
Lok
,
Louisa
Penicaut
,
Roksana
Ogrodowicz
,
Raveena
Prema
,
Simone
Kunzelmann
,
Chloe
Roustan
,
Ganka
Bineva‐todd
,
Saskia
Pieters
,
Francesca
Zappacosta
,
Alfred E.
Doherty
,
Isobel
Oram
,
Christelle
Soudy
,
Robert
Quinlan
,
Joanna
Redmond
,
Svend
Kjaer
,
David
House
,
Stephane
Mouilleron
,
Jacob T.
Bush
,
Benjamin
Schumann
Diamond Proposal Number(s):
[29040]
Open Access
Abstract: O-GalNAc (N-acetylgalactosaminyl) glycosylation is an abundant posttranslational modification in mammalian cells. Dysregulation of O-GalNAc glycosylation is implicated in cancer metastasis and immune evasion; however, our mechanistic understanding remains limited due to the lack of small-molecule tools. O-GalNAc biosynthesis depends heavily on the availability of UDP-GalNAc that is biosynthesised by the cytosolic enzyme UDP-galactose-4-epimerase (GalE). Knockout studies have demonstrated that loss of GalE severely impairs O-GalNAc glycosylation, positioning GalE as a promising enzymatic therapeutic target in oncology. Here, we present an efficient workflow that combines both covalent and high-throughput crystallographic non-covalent fragment screening with structure-based design to identify GalE inhibitors. Using these strategies, we discovered a ligandable pocket adjacent to a reactive tyrosine, enabling the development of a potent, “beyond cysteine” sulfonyl fluoride covalent inhibitor as well as a derived covalent alkyne probe. Structurally-enabled fragment screening methodologies yielded nanomolar non-covalent as well as covalent binders within no more than 22 elaborated compounds. Our work demonstrates synergism in next-generation delivery of chemical matter for GalE inhibition, with the broader potential for targeting non-cysteine residues in chemical biology and therapeutic applications.
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Apr 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25587]
Open Access
Abstract: Phosphoprotein phosphatase 1 (PP1) relies on association with PP1-interacting proteins (PIPs) to generate substrate-specific PIP/PP1 holoenzymes, but the lack of well-defined substrates has hindered elucidation of the mechanisms involved. We previously demonstrated that the Phactr1 PIP confers sequence specificity on the Phactr1/PP1 holoenzyme by remodelling the PP1 hydrophobic substrate groove. Phactr1 defines a group of ‘RVxF-ΦΦ-R-W’ PIPs that all interact with PP1 in a similar fashion. Here, we use a PP1-PIP fusion approach to address sequence specificity and identify substrates of the RVxF-ΦΦ-R-W family PIPs. We show that the four Phactr proteins confer identical sequence specificities on their holoenzymes. We identify the 4E-BP and p70 S6K translational regulators as substrates for the Neurabin/Spinophilin PIPs, implicated in neuronal plasticity, pointing to a role for their holoenzymes in mTORC1-dependent translational control. Biochemical and structural experiments show that in contrast to the Phactrs, substrate recruitment and catalytic efficiency of the PP1-Neurabin and PP1-Spinophilin fusions is primarily determined by substrate interaction with the PDZ domain adjoining their RVxF-ΦΦ-R-W motifs, rather than by recognition of the remodelled PP1 hydrophobic groove. Thus, even PIPs that interact with PP1 in a similar manner use different mechanisms to ensure substrate selectivity.
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Jun 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25587]
Open Access
Abstract: Peptidyl arginine deiminase 6 (PADI6) is vital for early embryonic development in mice and humans, yet its function remains elusive. PADI6 is less conserved than other PADIs and it is currently unknown whether it has a catalytic function. Here we have shown that human PADI6 dimerises like hPADIs 2-4, however, does not bind Ca2+ and is inactive in in vitro assays against standard PADI substrates. By determining the crystal structure of hPADI6, we show that hPADI6 is structured in the absence of Ca2+ where hPADI2 and hPADI4 are not, and the Ca-binding sites are not conserved. Moreover, we show that whilst the key catalytic aspartic acid and histidine residues are structurally conserved, the cysteine is displaced far from the active site centre and the hPADI6 active site pocket appears closed through a unique evolved mechanism in hPADI6, not present in the other PADIs. Taken together, these findings provide insight into how the function of hPADI6 may differ from the other PADIs based on its structure and provides a resource for characterising the damaging effect of clinically significant PADI6 variants.
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Aug 2024
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yakubu Princely
Abudu
,
Birendra
Kumar Shrestha
,
Wenxin
Zhang
,
Anthimi
Palara
,
Hanne Britt
Brenne
,
Kenneth Bowitz
Larsen
,
Deanna Lynn
Wolfson
,
Gianina
Dumitriu
,
Cristina Ionica
Øie
,
Balpreet Singh
Ahluwalia
,
Gahl
Levy
,
Christian
Behrends
,
Sharon A.
Tooze
,
Stephane
Mouilleron
,
Trond
Lamark
,
Terje
Johansen
Diamond Proposal Number(s):
[18566]
Abstract: Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.
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Aug 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Martina
Wirth
,
Stephane
Mouilleron
,
Wenxin
Zhang
,
Eva
Sjøttem
,
Yakubu
Princely Abudu
,
Ashish
Jain
,
Hallvard
Lauritz Olsvik
,
Jack-Ansgar
Bruun
,
Minoo
Razi
,
Harold B. J.
Jefferies
,
Rebecca
Lee
,
Dhira
Joshi
,
Nicola
O'Reilly
,
Terje
Johansen
,
Sharon A.
Tooze
Diamond Proposal Number(s):
[9826]
Open Access
Abstract: Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood.
We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic.
Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.
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Jun 2021
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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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Roman O.
Fedoryshchak
,
Magdalena
Přechová
,
Abbey
Butler
,
Rebecca
Lee
,
Nicola
O'Reilly
,
Helen R
Flynn
,
Ambrosius P
Snijders
,
Noreen
Eder
,
Sila
Ultanir
,
Stephane
Mouilleron
,
Richard
Treisman
Diamond Proposal Number(s):
[9826, 9826, 18566]
Open Access
Abstract: PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally-enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin aII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.
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Sep 2020
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8015]
Abstract: RPEL proteins, which contain the G-actin-binding RPEL motif, coordinate cytoskeletal processes with actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activating proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-actin complex, and show that G-actin contacts the RPEL motif and GAP domain sequences. G-actin inhibits ArhGAP12 GAP activity, and this requires the G-actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-actin assembly, invadopodia formation and experimental metastasis. In this setting, ArhGAP12 mutants defective for G-actin binding exhibit more effective downregulation of Rac GTP loading following HGF stimulation and enhanced inhibition of Rac-dependent processes, including invadopodia formation. Potentiation or disruption of the G-actin/ArhGAP12 interaction, by treatment with the actin-binding drugs latrunculin B or cytochalasin D, has corresponding effects on Rac GTP loading. The interaction of G-actin with RPEL-family rhoGAPs thus provides a negative feedback loop that couples Rac activity to actin dynamics.
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Jun 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13775]
Open Access
Abstract: Autophagy is an essential recycling and quality control pathway. Mammalian ATG8 proteins drive autophagosome formation and selective removal of protein aggregates and organelles by recruiting autophagy receptors and adaptors that contain a LC3-interacting region (LIR) motif. LIR motifs can be highly selective for ATG8 subfamily proteins (LC3s/GABARAPs), however the molecular determinants regulating these selective interactions remain elusive. Here we show that residues within the core LIR motif and adjacent C-terminal region as well as ATG8 subfamily-specific residues in the LIR docking site are critical for binding of receptors and adaptors to GABARAPs. Moreover, rendering GABARAP more LC3B-like impairs autophagy receptor degradation. Modulating LIR binding specificity of the centriolar satellite protein PCM1, implicated in autophagy and centrosomal function, alters its dynamics in cells. Our data provides new mechanistic insight into how selective binding of LIR motifs to GABARAPs is achieved, and elucidate the overlapping and distinct functions of ATG8 subfamily proteins.
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May 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Åsa Birna
Birgisdottir
,
Stephane
Mouilleron
,
Zambarlal
Bhujabal
,
Martina
Wirth
,
Eva
Sjøttem
,
Gry
Evjen
,
Wenxin
Zhang
,
Rebecca
Lee
,
Nicola
O’reilly
,
Sharon A.
Tooze
,
Trond
Lamark
,
Terje
Johansen
Diamond Proposal Number(s):
[9826, 13775]
Open Access
Abstract: Autophagosome formation depends on a carefully orchestrated interplay between membrane-associated protein complexes. Initiation of macroautophagy/autophagy is mediated by the ULK1 (unc-51 like autophagy activating kinase 1) protein kinase complex and the autophagy-specific class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1). The latter contains PIK3C3/VPS34, PIK3R4/VPS15, BECN1/Beclin 1 and ATG14 and phosphorylates phosphatidylinositol to generate phosphatidylinositol 3-phosphate (PtdIns3P). Here, we show that PIK3C3, BECN1 and ATG14 contain functional LIR motifs and interact with the Atg8-family proteins with a preference for GABARAP and GABARAPL1. High resolution crystal structures of the functional LIR motifs of these core components of PtdIns3K-C1were obtained. Variation in hydrophobic pocket 2 (HP2) may explain the specificity for the GABARAP family. Mutation of the LIR motif in ATG14 did not prevent formation of the PtdIns3K-C1 complex, but blocked colocalization with MAP1LC3B/LC3B and impaired mitophagy. The ULK-mediated phosphorylation of S29 in ATG14 was strongly dependent on a functional LIR motif in ATG14. GABARAP-preferring LIR motifs in PIK3C3, BECN1 and ATG14 may, via coincidence detection, contribute to scaffolding of PtdIns3K-C1 on membranes for efficient autophagosome formation.
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Mar 2019
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I02-Macromolecular Crystallography
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M. Teresa
Bertran
,
Stephane
Mouilleron
,
Yanxiang
Zhou
,
Rakhi
Bajaj
,
Federico
Uliana
,
Ganesan Senthil
Kumar
,
Audrey
Van Drogen
,
Rebecca
Lee
,
Jennifer J.
Banerjee
,
Simon
Hauri
,
Nicola
O’reilly
,
Matthias
Gstaiger
,
Rebecca
Page
,
Wolfgang
Peti
,
Nicolas
Tapon
Diamond Proposal Number(s):
[9826]
Open Access
Abstract: Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP (apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. Here we show that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. We solve the crystal structure of the human ASPP2/PP1 complex and show that ASPP2 recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1.
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Feb 2019
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