B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15868, 22906]
Abstract: In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGP synthase, IGPS) starts with a condensation step where the substrate’s carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation which restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. This step was since the 1960s considered to be mechanistically essential based on studies of the IGPS:PRAI fusion protein from Escherichia coli. Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a non-reactive substrate analogue, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate (PAdRP) – with approximately 1000' lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, out of which seven contain ligand, and one displays a previously unobserved conformation closer to the reactive state. One of the few non-conserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that, despite IGPS’s classification as a carboxylyase (i.e. decarboxylase), decarboxylation not a completely essential step in its catalysis.
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Sep 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Abstract: Centrioles are key eukaryotic organelles responsible for the formation of cilia and flagella, and for organising the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerisation of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organisation of further organelle components. A dimerisation interaction between SAS-6 N-terminal ‘head’ domains was previously shown to be essential for protein oligomerisation in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low molecular weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerisation. We demonstrate using nuclear magnetic resonance spectroscopy that a ligand from this family binds at the head domain dimerisation site of algae, nematode and human SAS-6 variants, but also that another ligand specifically recognises human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest ligand specificity derives from favourable Van der Waals interactions with a hydrophobic cavity at the dimerisation site.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Ribonucleotide reductase (RNR) is a central enzyme for DNA building block synthesis. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, electron microscopy, x-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral, but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.
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Sep 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Cecilia
Piergentili
,
Jennifer
Ross
,
Didi
He
,
Kelly J.
Gallagher
,
Will A.
Stanley
,
Laurène
Adam
,
C. Logan
Mackay
,
Arnaud
Basle
,
Kevin J.
Waldron
,
David J.
Clarke
,
Jon
Marles-wright
Diamond Proposal Number(s):
[9487]
Abstract: Encapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and neighboring secondary metal binding sites. We have previously identified a putative metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.
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Sep 2020
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800, 1993]
Abstract: Ticks, as blood sucking parasites, have developed a complex strategy to evade and suppress host immune responses during feeding. The crucial part of this strategy is expression of a broad family of salivary proteins, called Evasins, to neutralize chemokines responsible for cell trafficking and recruitment. However, structural information about Evasins is still scarce and little is known about the structural determinants of their binding mechanism to chemokines. Here, we studied the structurally uncharacterized Evasin-4, which neutralizes a broad range of CC-motif chemokines, including the chemokine CC-motif ligand 5 (CCL5) involved in atherogenesis. Crystal structures of Evasin-4 and E66S CCL5—an obligatory dimeric variant of CCL5—were determined to a resolution of 1.3-1.8 Å. The Evasin-4 crystal structure revealed an L-shaped architecture formed by an N- and C-terminal subdomain consisting of eight β-strands and an α-helix that adopts a substantially different position compared to closely related Evasin-1. Further investigation into E66S CCL5/Evasin-4 complex formation with NMR spectroscopy showed that residues of the N-terminus are involved in binding to CCL5. The peptide derived from the N-terminal region of Evasin-4 possessed nM affinity to CCL5 and inhibited CCL5 activity in monocyte migration assays. This suggests that Evasin-4 derivatives could be used as starting point for the development of anti-inflammatory drugs.
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Aug 2020
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I04-Macromolecular Crystallography
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Abstract: Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly-identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling the plant tumor development. Here we report the structural basis of SghR, a LacI-type transcription factor and highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA, and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling crosstalk during Agrobacterium infection, our results pave the way for structure-based inducer analogue design, which has potential applications for agricultural industry.
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Jul 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19036]
Abstract: Since the advent of protein crystallography, atomic-level macromolecular structures have provided a basis to understand biological function. Enzymologists use detailed structural insights on ligand coordination, interatomic distances and positioning of catalytic amino acids to rationalize the underlying electronic reaction mechanisms. Often the proteins in question catalyze redox reactions using metal cofactors that are explicitly intertwined with their function. In these cases, the exact nature of the coordination sphere and the oxidation state of the metal is of utmost importance. Unfortunately, the redox active nature of metal cofactors makes them especially susceptible to photoreduction, meaning that information obtained by photoreducing X-ray sources about the environment of the cofactor are the least trustworthy part of the structure. In this work we directly compare the kinetics of photoreduction of six different heme protein crystal species at by X-ray radiation. We show that a dose of approximately 40 kGy already yields 50% ferrous iron in a heme protein crystal. We also demonstrate that the kinetics of photoreduction are completely independent from variables unique to the different samples tested. The photoreduction-induced structural rearrangements around the metal cofactors have to be considered when biochemical data of ferric proteins are rationalized by constraints derived from crystal structures of reduced enzymes.
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Jul 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: The human gut symbiont Ruminococcus gnavus scavenges host‐derived N‐acetylneuraminic acid (Neu5Ac) from mucins, by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D nuclear magnetic resonance (NMR), we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-DANA intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram negative and Gram positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionisation spray mass spectrometry (ESI-MS) that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolise 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry (DSF) analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli was dependent on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.
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Jul 2020
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I03-Macromolecular Crystallography
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Mark J
Burton
,
Joel
Cresser-brown
,
Morgan
Thomas
,
Nicola
Portolano
,
Jaswir
Basran
,
Samuel L.
Freeman
,
Hanna
Kwon
,
Andrew R.
Bottrill
,
Manuel J
Llansola-portoles
,
Andrew A
Pascal
,
Rebekah
Jukes-jones
,
Tatyana
Chernova
,
Ralf
Schmid
,
Noel W.
Davies
,
Nina M.
Storey
,
Pierre
Dorlet
,
Peter C. E.
Moody
,
John S
Mitcheson
,
Emma L.
Raven
Diamond Proposal Number(s):
[14692]
Abstract: The ether-à-go-go (EAG) family of voltage gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the amino-terminus, known as the eag-domain, consisting of a PAS domain capped by a short sequence containing an amphipathic helix (Cap-domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pull-down assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in E.coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.
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Jul 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[5133]
Abstract: Hepatic abundance of the Low-Density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL-cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase Inducible Degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (F for 4.1 protein, E for ezrin, R for radixin and M for moesin) domain, responsible for substrate recognition and plasma-membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homo-dimerization. As IDOL is a putative lipid-lowering drug-target we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high auto-ubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 tri-lobed FERM domain structure but show that the F3c subdomain orientation obscures the target binding site. To substantiate this finding, we analyzed the full length FERM domain and a series of truncated FERM constructs by small angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.
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Jul 2020
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