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Gerard H. M.
Huysmans
,
Nathan
Chan
,
Jocelyn M.
Baldwin
,
Vincent L. G.
Postis
,
Svetomir B.
Tzokov
,
Sarah E.
Deacon
,
Sylvia Y. M.
Yao
,
James D.
Young
,
Michael J.
Mcpherson
,
Per A.
Bullough
,
Stephen A.
Baldwin
Abstract: Urea is exploited as a nitrogen source by bacteria, and its breakdown products, ammonia and bicarbonate, are employed to counteract stomach acidity in pathogens such as Helicobacter pylori. Uptake in the latter is mediated by UreI, a UAC (urea amide channel) family member. In the present paper, we describe the structure and function of UACBc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea, but also to other small amides. CD and IR spectroscopy revealed a structure comprising mainly ?-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of seven TM (transmembrane) helices, with a cytoplasmic C-terminus. In detergent, UACBc exists largely as a hexamer, as demonstrated by both cross-linking and size-exclusion chromatography. A 9 Å (1 Å=0.1 nm) resolution projection map obtained by cryo-electron microscopy of two-dimensional crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, with the resultant model enabling identification of residues likely to contribute to channel function.
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Jul 2012
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[14794]
Open Access
Abstract: The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid β-oxidation cycle. Two TFEs are present in Escherichia coli , EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane bound complex. Systematic enzyme kinetic studies show that anEcTFE has preference for medium and long chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.
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Jun 2019
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
Data acquisition
Diagnostics
Health Physics
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa , known as E-type colicins and S-type pyocins, respectively, bound to their specific immunity (Im) proteins. First, we report the 3.2 Å structure of the DNase colicin ColE9 in complex with its ultra-high affinity immunity protein, Im9. In contrast to Im3, which when bound to the ribonuclease (rRNase) domain of the homologous colicin ColE3 makes contact with the translocation (T-) domain of the toxin, we find that Im9 makes no such contact and only interactions with the ColE9 cytotoxic domain are observed. Second, we report small angle X-ray scattering (SAXS) data for two S-type DNase pyocins, S2 and AP41, into which are fitted recently determined X-ray structures for isolated domains. We find that DNase pyocins and colicins are both highly elongated molecules even though the order of their constituent domains differs. We discuss the implications of these architectural similarities and differences in the context of the translocation mechanism of protein antibiotics through the cell envelope of Gram-negative bacteria.
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Jul 2016
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
|
Open Access
Abstract: The group A p21-activated kinases (PAKs) exist in an auto-inhibited form until activated by GTPase binding and auto-phosphorylation. In the auto-inhibited form a regulatory domain binds to the kinase domain blocking the binding of substrates, and CDC42 or Rac binding to the regulatory domain relieves this auto-inhibition allowing auto-phosphorylation on the kinase domain activation loop. We have determined the crystal structure of the PAK3 catalytic domain and by small angle X-ray scattering the solution-phase structures of full-length inactive PAK1 and PAK3. The structures reveal a compact but elongated molecular shape that demonstrates that, together with multiple independent biophysical measurements and in contrast to previous assumptions, group A PAKs are monomeric both before and after activation, consistent with an activation mechanism of cis -autoinhibition and initial cis -autophosphorylation, followed by transient dimerization to allow trans- autophosphorylation for full activation, yielding a monomeric active PAK protein.
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Apr 2019
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[8421]
Abstract: Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5:5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB-BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.
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Sep 2017
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9537]
Abstract: Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) signaling family and has a very broad biological role in development. Its signaling is regulated by many effectors: transmembrane proteins, membrane attached proteins and soluble secreted antagonists such as Gremlin-1. Very little is known about the molecular mechanism by which Gremlin-1 and other DAN family proteins inhibit BMP signaling. We analyzed the interaction of Gremlin-1 with BMP-2 using a range of biophysical techniques, and used mutagenesis to map the binding site on BMP-2. We have also determined the crystal structure of Gremlin-1, revealing a similar conserved dimeric structure as has been seen in other DAN family inhibitors. Measurements using biolayer interferometry indicate that Gremlin-1 and BMP-2 can form larger complexes, beyond the expected 1:1 stoichiometry of dimers, forming oligomers that assemble in alternating fashion. These results suggest that inhibition of BMP-2 by Gremlin-1 occurs by a mechanism that is distinct from other known inhibitors such as Noggin and Chordin and we propose a novel model of BMP-2/Gremlin-1 interaction yet not seen among any BMP antagonists, and cannot rule out that several different oligomeric states could be found, depending on the concentration of the two proteins.
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Apr 2016
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[14894, 16440]
Abstract: The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism, and small angle neutron scattering, we have studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered, with a mobile C-terminal domain and no changes in secondary structure but increases in the radius of gyration on complex formation. Comparison of wild type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.
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Jul 2017
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[15094, 17698, 19120]
Open Access
Abstract: The light reactions of photosynthesis are hosted and regulated by the chloroplast thylakoid membrane (TM) — the central structural component of the photosynthetic apparatus of plants and algae. The two-dimensional and three-dimensional arrangement of the lipid–protein assemblies, aka macroorganisation, and its dynamic responses to the fluctuating physiological environment, aka flexibility, are the subject of this review. An emphasis is given on the information obtainable by spectroscopic approaches, especially circular dichroism (CD). We briefly summarise the current knowledge of the composition and three-dimensional architecture of the granal TMs in plants and the supramolecular organisation of Photosystem II and light-harvesting complex II therein. We next acquaint the non-specialist reader with the fundamentals of CD spectroscopy, recent advances such as anisotropic CD, and applications for studying the structure and macroorganisation of photosynthetic complexes and membranes. Special attention is given to the structural and functional flexibility of light-harvesting complex II in vitro as revealed by CD and fluorescence spectroscopy. We give an account of the dynamic changes in membrane macroorganisation associated with the light-adaptation of the photosynthetic apparatus and the regulation of the excitation energy flow by state transitions and non-photochemical quenching.
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Oct 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
Data acquisition
Detectors
Diagnostics
Health Physics
|
Diamond Proposal Number(s):
[6386]
Abstract: α-actinin 2 (ACTN2) is the only muscle isoform of α-actinin expressed in cardiac muscle. Mutations in this protein have been implicated in mild to moderate forms of hypertrophic cardiomyopathy (HCM). We have investigated the effects of two mutations identified from HCM patients; A119T and G111V, on the secondary and tertiary structure of a purified actin binding domain of ACTN2 by circular dichroism and X-ray crystallography, and show small but distinct changes for both mutations. We also find that both mutants have reduced F-actin binding affinity, although the differences are not significant. The full length mEos2 tagged protein expressed in adult cardiomyocytes shows that both mutations additionally affect Z-disc localisation and dynamic behaviour. Overall, these two mutations have small effects on structure, function and behaviour, which may contribute to a mild phenotype for this disease.
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Aug 2016
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I02-Macromolecular Crystallography
|
Open Access
Abstract: The interaction between streptavidin and biotin is one of the strongest non-covalent interactions in nature. Streptavidin is a widely used tool and a paradigm for protein-ligand interactions. We recently developed a streptavidin mutant, termed traptavidin, possessing 10-fold lower off-rate for biotin, with increased mechanical and thermal stability. Here, we determined crystal structures of apo-traptavidin and biotin-traptavidin at 1.5 Å resolution. In apo-streptavidin the L3/4 loop, near biotins valeryl tail, is typically disordered and open, but closes upon biotin binding. In contrast, this L3/4 loop was shut in both apo-traptavidin and biotin-traptavidin. The reduced flexibility of L3/4 and decreased conformational change on biotin binding provide an explanation for traptavidins reduced biotin off-rate and on-rate. The L3/4 loop includes Ser-45, which forms a hydrogen bond to biotin consistently in traptavidin but erratically in streptavidin. Reduced breakage of the biotin:Ser-45 hydrogen bond in traptavidin is likely to inhibit the initiating event in biotins dissociation pathway. We generated a traptavidin with 1 biotin binding site rather than 4, which showed a similarly slow off-rate, demonstrating that traptavidins slow off-rate was governed by intra-subunit effects. Understanding the structural features of this tenacious interaction may assist design of even stronger affinity tags and inhibitors.
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Jan 2011
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