I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[35324]
Open Access
Abstract: Prokaryotic type III CRISPR systems signal infection by generating cyclic oligoadenylate (cOA) second messengers, which activate defence proteins allosterically, providing immunity. cOA molecules are typically degraded by extrinsic, stand-alone ring nuclease (RN) enzymes with phosphodiesterase activity, or by the intrinsic RN activity of the effectors themselves. Viruses and plasmids also encode RNs, which can function as anti-CRISPRs (Acr). Eight different families of extrinsic RNs are currently known. Here, we report the structural and biochemical analysis of one of these families: Csx15. We show that Csx15 is a dimeric protein of the CARF (CRISPR associated Rossmann fold) superfamily with the ability to bind cyclic tetra-adenylate (cA4) molecules in a shared binding site formed by the head-to-tail stacking of dimers in a filament conformation. Some family members are non-enzymatic, relying on the sequestration (sponging) of cA4 to regulate the host immune response, while others act as canonical RNs, slowly degrading cA4.
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Mar 2026
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I03-Macromolecular Crystallography
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David
Carling
,
Shu-Yang
Chen
,
Jane
Bennett
,
Naveenan
Navaratnam
,
Rebeca
Fiadeiro
,
Angela
Woods
,
Alex
Montoya
,
Pavel
Shliaha
,
Simone
Kunzelmann
,
Steven A.
Howell
,
Shahid
Mehmood
,
Andrew G.
Purkiss
,
Jon R.
Wilson
,
Steven J.
Gamblin
Diamond Proposal Number(s):
[18566]
Abstract: AMP-activated protein kinase (AMPK) plays an important role in maintaining energy homeostasis in mammals. AMPK is heterotrimer of an α catalytic subunit, and two regulatory subunits, β and γ. In mammals, each subunit has different isoforms (α1/α2, β1/β2, and γ1/γ2/γ3) encoded by separate genes leading to the potential expression of 12 AMPK complexes. Here we show that AMPK containing the long forms of γ2 (γ2a, encoding a protein of 569 amino acids, and γ2c, 525 amino acids) bind to 14-3-3. In contrast to AMPK containing the short form of γ2 (γ2b, 328 amino acids), bacterial expression of AMPK containing the long forms of g2 requires co-expression with 14-3-3 and prior phosphorylation of Thr172 within the α subunit. AMPKγ2-14-3-3 complexes have reduced activity compared to AMPKγ1 or AMPKγ2b but retain allosteric activation by AMP and the AMPK activator, 991. We found that two predicted 14-3-3 binding sites within γ2a (T97 and S122) were phosphorylated in the bacterially expressed AMPK complex. Furthermore, we show that a peptide spanning these two phosphorylated sites binds to 14-3-3 in vitro and determined the crystal structure of this 14-3-3-peptide co-complex. These results indicate that 14-3-3 binds to the N-terminal region of γ2a/c, reducing the activity of AMPK relative to AMPKγ1 and AMPKγ2b. Our findings reveal a new mode of regulation of AMPK containing the long forms of γ2. Whilst the biological significance of 14-3-3 binding to AMPKγ2a/c complexes remains to be determined, our studies provide the starting point to begin to address this issue.
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Mar 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[35324]
Open Access
Abstract: Type III CRISPR systems typically generate cyclic oligoadenylate second messengers such as cyclic tetra-adenylate (cA4) on detection of foreign RNA. These activate ancillary effector proteins which elicit a diverse range of immune responses. The Calp (CRISPR associated Lon protease) system elicits a transcriptional response to infection when CalpL (Calp Lon protease) binds cA4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS (Calp Sigma factor) for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2 (second anti-CRISPR of type III systems), which they use to degrade cA4. AcrIII-2 dimers sandwich cA4, degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA4 activated effectors in vitro. This is the first example of a virally encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.
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Nov 2025
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I04-Macromolecular Crystallography
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Tom D.
Bunney
,
Hunter G.
Nyvall
,
Calum
Macrae
,
Damjan
Lalović
,
Ashley
Gregory
,
Kyle I. P.
Le Huray
,
Nikita
Harvey
,
Nikos
Pinotsis
,
Antreas C.
Kalli
,
Christopher A.
Waudby
,
John E.
Burke
,
Matilda
Katan
Diamond Proposal Number(s):
[30393]
Open Access
Abstract: Phospholipase C gamma (PLCγ) enzymes are key components of intracellular signal transduction processes and are involved in disease development, including immune dysregulation, specific cancer types and neurodegeneration. Although recognised as important targets for intervention, validated pharmacological tools are lacking. Here, we demonstrate that inhibitory nucleotides bind directly to an allosteric site at the interface between the PLC-core and regulatory-array unique for PLCγ, underlying their specificity for the PLCγ family. This binding site overlaps with the PLCγ autoinhibitory interface, suggesting that the inhibitory impact of nucleotides involves stabilisation of autoinhibition. We have also analysed disease-linked variants of PLCγ1 and PLCγ2 to show that multiple mechanisms could underpin their gain-of-function phenotype. While the sensitivity of these variants to physiological nucleotide inhibition is reduced, we identified artificial nucleotide compounds that can inhibit such variants not only in vitro but also in cell-based assays. Therefore, our findings suggest a route for development of isozyme specific PLCγ inhibitors allowing further studies of their roles in health and disease.
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Oct 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[39391]
Open Access
Abstract: Mutations in the T-cell receptor signalling pathway have been identified in patients with adult T-cell leukaemia/lymphoma (ATLL) and one of the most frequently observed targets of these mutations is protein kinase C beta (PKCb). Here we have characterised the most frequent mutation in PKCb (D427N) addressing the issue of gain/loss of function, neomorphic change, assessing the impact of mutation in vivo, in cells, biochemically and structurally. It is concluded that this mutation is a gain-of-function, activating mutation that confers an altered substrate specificity on this protein kinase. In a constitutive knock-in mouse model this activated allele induces splenomegaly associated with extramedullary haematopoiesis. Pharmacologically, the D427N mutant protein displays poor sensitivity to established PKCb inhibitors, necessitating development of bespoke therapeutics for any ATLL intervention through this target. Such efforts could be guided by the availability the D427N mutant-ruboxistaurin structure presented here.
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Oct 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Pseudomonas aeruginosa PA01 is one of the major causes of disease persistence and mortality in patients with lung pathologies, relying on various host metabolites as carbon and energy sources for growth. The ict-ich-ccl operon (pa0878, pa0882 and pa0883) in PAO1 is required for growth on the host molecule itaconate, a C5-dicarboxylate. However, it is not known how itaconate is taken up into P. aeruginosa. Here, we demonstrate that a genetically linked tripartite ATP-independent periplasmic (TRAP) transporter (pa0884-pa0886), which is homologous to the known C4-dicarboxylate-binding TRAP system, is essential for growth on itaconate, but not for the closely related C4-dicarboxylate succinate. Using tryptophan fluorescence spectroscopy, we demonstrate that the substrate-binding protein (SBP), IctP (PA0884), binds itaconate but still retains higher affinity for the related C4-dicarboxylates. The structures of IctP bound to itaconate (1.80 Å) and succinate (1.75 Å) revealed an enclosed ligand-binding pocket with ion pairing interactions with the ligand carboxylates. The C2 methylene group that is the distinguishing feature of itaconate compared with succinate is accommodated by a unique change in the IctP-binding site from a Leu to Val, which distinguishes it from closely related C4-dicarboxylate-binding SBPs. Together, these data suggest that this transporter, which we name IctPQM, has duplicated from a canonical C4-dicarboxylate transporter, and its evolution towards itaconate specificity enables this pathogen to now access a key metabolite for persistence in the host.
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Sep 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: In Leishmania parasites, as for their hosts, the ubiquitin proteasome system is important for cell viability. As part of a systematic gene deletion study, it was discovered that four cysteine protease type deubiquitinases (DUBs) are essential for parasite survival in the promastigote stage, including DUB16. Here we have purified and characterised recombinant DUB16 from Leishmania donovani, which belongs to the ubiquitin C-terminal hydrolase (UCH) family. DUB16 efficiently hydrolyses C-terminal aminocoumarin and rhodamine conjugates of ubiquitin consistent with proposed cellular roles of UCH-type DUBs in regenerating free monomeric ubiquitin from small molecule ubiquitin adducts arising from adventitious metabolic processes. The crystal structure of DUB16 reveals a typical UCH-type deubiquitinase fold, and a relatively short and disordered crossover loop that appears to restrict access to the catalytic cysteine. At close to stoichiometric enzyme to substrate ratios, DUB16 exhibits deubiquitinase activity towards diubiquitins linked through isopeptide bonds between Lys11, Lys48 or Lys63 residues of the proximal ubiquitin and the C-terminus of the distal ubiquitin. With 100-1000-fold higher turnover rates, DUB16 cleaves the ubiquitin-ribosomal L40 fusion protein to give the mature products. A DUB-targeting cysteine-reactive cyanopyrrolidine compound, IMP-1710, inhibits DUB16 activity. IMP-1710 was shown in promastigote cell viability assays to have parasite killing activity with EC50 values of 1-2 M, comparable to the anti-leishmanial drug, miltefosine. L. mexicana parasites engineered to overproduce DUB16 showed a modest increase in resistance to IMP-1710, providing evidence that IMP-1710 inhibits DUB16 in vivo. While it is highly likely that IMP-1710 has additional targets, these results suggest that DUB16 is a potential target for the development of new anti-leishmanial compounds.
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Jun 2025
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[29074, 35120]
Open Access
Abstract: The adaptor protein, Speckle-type BTB/POZ protein (SPOP), recruits substrates to the cullin-3-subclass of E3 ligase for selective protein ubiquitylation. The Myddosome protein, Myeloid differentiation primary response 88 (MyD88), is ubiquitylated by the SPOP-based E3 ligase to negatively regulate immune signaling, however, the sequence rules for SPOP-mediated substrate engagement and degradation are not fully understood. Here, we show that MyD88 interacts with SPOP through a long degron that contains the established SPOP-binding consensus and an N-terminal site that we name the Q-motif. Based on sequence similarity to MyD88, we show that additional substrates, including Steroid receptor coactivator-3 (SRC-3), SET domain-containing protein 2 (SETD2) and Caprin1, engage SPOP in this manner. We show that the Q-motif is a critical determinant of these interactions in mammalian cells and determine X-ray crystal structures that show the molecular basis of SPOP associations with these proteins. These studies reveal a new consensus sequence for substrate-binding to SPOP that is necessary for substrate ubiquitylation, thus expanding the sequence rules required for SPOP-mediated E3 ligase substrate recognition.
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Mar 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32736]
Open Access
Abstract: The sulfosugar sulfoquinovose (SQ) is catabolized through the sulfoglycolytic Entner-Doudoroff pathway, beginning with the oxidation of SQ to sulfogluconolactone by SQ dehydrogenase. We present a comprehensive structural and kinetic characterization of Pseudomonas putida SQ dehydrogenase (PpSQDH). PpSQDH is a tetrameric enzyme belonging to the short-chain dehydrogenase/reductase (SDR) superfamily with a strong preference for NAD+ over NADP+. Kinetic analysis revealed a rapid equilibrium ordered mechanism in which the NAD+ cofactor is the first substrate to bind, and NADH is the last product to dissociate. Structural studies revealed a homotetrameric structure in solution and crystals, involving cross-subunit interactions in which the C-terminus residue (Gln260) inserts into the diagonally opposite subunit to form part of the second shell of residues lining the active site. Complexes of PpSQDH with SQ or NAD+ provide insight into the recognition of SQ and together with the kinetic analysis allow the proposal of a catalytic reaction mechanism. Our findings illuminate the mechanism of SQ degradation and the evolution of the SDR superfamily for organosulfonate catabolism.
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Jan 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19248]
Open Access
Abstract: The PDZ (Postsynaptic density protein-95[PSD-95]/Discs-large) domain, prevalent as a recognition module, has attracted significant attention given its ability to specifically recognize ligands with consensus motifs (also termed PDZ binding motifs [PBMs]). PBMs typically bear a C-terminal carboxylate as a recognition handle and have been extensively characterised, whilst internal ligands are less well known. Here we characterize a short linear motif (SLiM) – EESTSFQGP – as an internal PBM based on its strong binding affinity towards the SHANK1 PDZ domain (SHANK1656-762 hereafter referred to as SHANK1). Using the acetylated analogue Ac-EESTSFQGP-CONH2 as a competitor for the interaction of SHANK1 with FAM-Ahx-EESTSFQGP-CONH2 or a typical fluorophore-labelled C-terminal PBM – GKAP – FITC-Ahx-EAQTRL-COOH – the internal SLiM was demonstrated to show comparable low-micromolar IC50 by competition fluorescent anisotropy (FA). To gain further insight on the internal ligand interaction at the molecular level, we obtained the X-ray co-crystal structure of the Ac-EESTSFQGP-CONH2/SHANK1 complex and compared this to the Ac-EAQTRL-COOH/SHANK1 complex. The crystallographic studies reveal that the SHANK1 backbones for the two interactions overlap significantly. The main structural differences were shown to result from the flexible loops which reorganise to accommodate the two PBMs with distinct lengths and terminal groups. In addition, the two C-terminal residues Gly and Pro in Ac-EESTSFQGP-CONH2 were shown not to participate in interaction with the target protein, implying further truncation and structural modification using peptidomimetic approaches on this sequence may be feasible. Taken together, the SLiM Ac-EESTSFQGP-CONH2 holds potential as an internal ligand for targeting SHANK1.
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Jun 2024
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