I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19951, 26302, 14794, 10291]
Open Access
Abstract: The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2β2 tetrameric enzyme. The α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) activity. Enzyme kinetic data reported here show that medium and long chain enoyl-CoA molecules are preferred substrates for MtTFE. Modelling studies indicate how the linear medium and long acyl chains of these substrates can bind to each of the active sites. In addition, crystallographic binding studies have identified three new CoA binding sites which are different from the previously known CoA binding sites of the three TFE active sites. Structure comparisons provide new insights into the properties of ECH, HAD and KAT active sites of MtTFE. The interactions of the adenine moiety of CoA with loop-2 of the ECH active site cause a conformational change of this loop by which a competent ECH active site is formed. The NAD+ binding domain (domain C) of the HAD part of MtTFE has only a few interactions with the rest of the complex and adopts a range of open conformations, whereas the A-domain of the ECH part is rigidly fixed with respect to the HAD part. Two loops, the CB1-CA1 region and the catalytic CB4-CB5 loop, near the thiolase active site and the thiolase dimer interface, have high B-factors. Structure comparisons suggest that a competent and stable thiolase dimer is formed only when complexed with the α-chains, highlighting the importance of the assembly for the proper functioning of the complex.
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Sep 2021
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Ed
Daniel
,
Mirko M.
Maksimainen
,
Neil
Smith
,
Ville
Ratas
,
Ekaterina
Biterova
,
Sudarshan N.
Murthy
,
M. Tanvir
Rahman
,
Tiila-Riikka
Kiema
,
Shruthi
Sridhar
,
Gabriele
Cordara
,
Subhadra
Dalwani
,
Rajaram
Venkatesan
,
Jaime
Prilusky
,
Orly
Dym
,
Lari
Lehtio
,
M. Kristian
Koski
,
Alun W.
Ashton
,
Joel L.
Sussman
,
Rikkert K.
Wierenga
Open Access
Abstract: The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank.
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Feb 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26302, 24732]
Abstract: The peroxisomal multifunctional enzyme type 1 (MFE1) catalyzes two successive reactions in the β-oxidation cycle: the 2E-enoyl-CoA hydratase (ECH) and NAD+-dependent 3S-hydroxyacyl-CoA dehydrogenase (HAD) reactions. MFE1 is a monomeric enzyme that has five domains. The N-terminal part (domains A and B) adopts the crotonase fold and the C-terminal part (domains C, D and E) adopts the HAD fold. A new crystal form of MFE1 has captured a conformation in which both active sites are noncompetent. This structure, at 1.7 Å resolution, shows the importance of the interactions between Phe272 in domain B (the linker helix; helix H10 of the crotonase fold) and the beginning of loop 2 (of the crotonase fold) in stabilizing the competent ECH active-site geometry. In addition, protein crystallographic binding studies using optimized crystal-treatment protocols have captured a structure with both the 3-ketodecanoyl-CoA product and NAD+ bound in the HAD active site, showing the interactions between 3-ketodecanoyl-CoA and residues of the C, D and E domains. Structural comparisons show the importance of domain movements, in particular of the C domain with respect to the D/E domains and of the A domain with respect to the HAD part. These comparisons suggest that the N-terminal part of the linker helix, which interacts tightly with domains A and E, functions as a hinge region for movement of the A domain with respect to the HAD part.
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Dec 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14794]
Abstract: Degradation of fatty acids by the β-oxidation pathway results in the formation of acetyl-CoA which enters the TCA cycle for the production of ATP. In E. coli, the last three steps of the β-oxidation are catalyzed by two heterotetrameric α2β2 enzymes namely the aerobic trifunctional enzyme (EcTFE) and the anaerobic TFE (anEcTFE). The α-subunit of TFE has 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities whereas the β-subunit is a thiolase with 3-ketoacyl-CoA thiolase (KAT) activity. Recently, it has been shown that the two TFEs have complementary substrate specificities allowing for the complete degradation of long chain fatty acyl-CoAs into acetyl-CoA under aerobic conditions. Also, it has been shown that the tetrameric EcTFE and anEcTFE assemblies are similar to the TFEs of Pseudomans fragi and human, respectively. Here the properties of the EcTFE subunits are further characterized. Strikingly, it is observed that when expressed separately, EcTFE-α is a catalytically active monomer whereas EcTFE-β is inactive. However, when mixed together active EcTFE tetramer is reconstituted. The crystal structure of the EcTFE-α chain is also reported, complexed with ATP, bound in its HAD active site. Structural comparisons show that the EcTFE hydratase active site has a relatively small fatty acyl tail binding pocket when compared to other TFEs in good agreement with its preferred specificity for short chain 2E-enoyl-CoA substrates. Furthermore, it is observed that millimolar concentrations of ATP destabilize the EcTFE complex, and this may have implications for the ATP-mediated regulation of β-oxidation in E. coli.
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Mar 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[14794]
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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Abstract: The SCP2-thiolase (type-1) functions in the vertebrate peroxisomal, bile acid synthesis pathway, converting 24-keto-THC-CoA and CoA into choloyl-CoA and propionyl-CoA. This conversion concerns the β-oxidation chain shortening of the steroid fatty acyl moiety of 24-keto-THC-CoA. This class of dimeric thiolases has previously been poorly characterized. High resolution crystal structures of the zebrafish SCP2-thiolase (type-1) now reveal an open catalytic site, shaped by residues of both subunits. The structure of its non-dimerised monomeric form has also been captured in the obtained crystals. Four loops at the dimer interface adopt very different conformations in the monomeric form. These loops also shape the active site and their structural changes explain why a competent active site is not present in the monomeric form. Native mass spectrometry studies confirm that the zebrafish SCP2-thiolase (type-1) as well as its human homologue are weak transient dimers in solution. The crystallographic binding studies reveal the mode of binding of CoA and octanoyl-CoA in the active site, highlighting the conserved geometry of the nucleophilic cysteine, the catalytic acid/base cysteine and the two oxyanion holes. The dimer interface of SCP2-thiolase (type-1) is equally extensive as in other thiolase dimers, however it is more polar than any of the corresponding interfaces, which correlates with the notion that the enzyme forms a weak transient dimer. The structure comparison of the monomeric and dimeric forms suggests functional relevance of this property. These comparisons provide also insight into the structural rearrangements that occur when the folded inactive monomers assemble into the mature dimer.
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Dec 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Abstract: Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5-fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA. Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP-acetoacetyl-CoA (1.90 Å) and TbSLP-malonyl-CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl-CoA tightly (Kd 90 µM), acetoacetyl-CoA moderately (Kd 0.9 mM) and acetyl-CoA and CoA very weakly. TbSLP possesses low malonyl-CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism.
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Aug 2016
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I03-Macromolecular Crystallography
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Abstract: The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using an Escherichia coli L-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of the Escherichia coli selection strain in medium supplemented with 40 mM L-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.
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Jun 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: The type III secretion system (T3SS) is required for the virulence of many gram-negative bacterial human pathogens. It is composed of several structural proteins, forming the secretion needle and its basis, the basal body. In Chlamydia spp., the T3SS inner membrane ring (IM-ring) of the basal body is formed by the periplasmic part of CdsD (outer ring) and CdsJ (inner ring). Here we describe the crystal structure of the C-terminal, periplasmic part of CdsD, not including the last 60 residues. Two crystal forms were obtained, grown in three different crystallization conditions. In both crystal forms there is one molecule per asymmetric unit adopting a similar extended structure. The structures consist of three periplasmic domains (PDs) of similar αββαβ topology as seen also in the structures of the homologous PrgH (Salmonella typhimurium) and YscD (Yersinia enterocolitica). Only in the C2 crystal form, there is a C-terminal additional helix after the PD3 domain. The relative orientation of the three subsequent CdsD PD domains with respect to each other is more extended than in PrgH but less extended than in YscD. Small-angle X-ray scattering data show that also in solution this CdsD construct adopts the same elongated shape. In both crystal forms the CdsD molecules are packed in a parallel fashion, using translational crystallographic symmetry. The most extensive crystal contacts are preserved in both crystal forms, suggesting a possible mode of assembly of the CdsD periplasmic part into a 24-mer complex forming the outer ring of the IM-ring of the T3SS.
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May 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Δ3,Δ2-Enoyl-CoA isomerases (ECIs) catalyze the shift of a double bond from 3Z- or 3E-enoyl-CoA to 2E-enoyl-CoA. ECIs are members of the crotonase superfamily. The crotonase framework is used by many enzymes to catalyze a wide range of reactions on acyl-CoA thioesters. The thioester O atom is bound in a conserved oxyanion hole. Here, the mode of binding of acyl-CoA substrate analogues to peroxisomal Saccharomyces cerevisiae ECI (ScECI2) is described. The best defined part of the bound acyl-CoA molecules is the 3′,5′-diphosphate-adenosine moiety, which interacts with residues of loop 1 and loop 2, whereas the pantetheine part is the least well defined. The catalytic base, Glu158, is hydrogen-bonded to the Asn101 side chain and is further hydrogen-bonded to the side chain of Arg100 in the apo structure. Arg100 is completely buried in the apo structure and a conformational change of the Arg100 side chain appears to be important for substrate binding and catalysis. The oxyanion hole is formed by the NH groups of Ala70 (loop 2) and Leu126 (helix 3). The O atoms of the corresponding peptide units, Gly69 O and Gly125 O, are both part of extensive hydrogen-bond networks. These hydrogen-bond networks are a conserved feature of the crotonase oxyanion hole and their importance for catalysis is discussed.
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Nov 2015
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