I24-Microfocus Macromolecular Crystallography
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Christa
Litschko
,
Valerio
Di Domenico
,
Julia
Schultz
,
Sizhe
Li
,
Olga G.
Ovchinnikova
,
Thijs
Voskuilen
,
Andrea
Bethe
,
Javier O.
Cifuente
,
Alberto
Marina
,
Insa
Budde
,
Tim A.
Mast
,
Małgorzata
Sulewska
,
Monika
Berger
,
Falk F. R.
Buettner
,
Todd L.
Lowary
,
Chris
Whitfield
,
Jeroen D. C.
Codée
,
Mario
Schubert
,
Marcelo E.
Guerin
,
Timm
Fiebig
Diamond Proposal Number(s):
[28360]
Open Access
Abstract: Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules.
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Jul 2024
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9475]
Open Access
Abstract: Inositol phosphates and their pyrophosphorylated derivatives are responsive to the phosphate supply and are agents of phosphate homeostasis and other aspects of physiology. It seems likely that the enzymes that interconvert these signals work against the prevailing milieu of mixed populations of competing substrates and products. The synthesis of inositol pyrophosphates is mediated in plants by two classes of ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to inositol pyrophosphate synthesis and turnover in plants is incomplete: the absence of nucleotide in published crystal structures limits the explanation of phosphotransfer reactions, and little is known of the affinity of potential substrates and competitors for ITPK1. Herein, we describe a complex of ADP and StITPK1 at 2.26 Å resolution and use a simple fluorescence polarization approach to compare the affinity of binding of diverse inositol phosphates, inositol pyrophosphates, and analogues. By simple HPLC, we reveal the novel catalytic capability of ITPK1 for different inositol pyrophosphates and show Ins(3,4,5,6)P4 to be a potent inhibitor of the inositol pyrophosphate-synthesizing activity of ITPK1. We further describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates.
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Dec 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Å resolution that along with description of the enantiospecificity of the enzyme affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a Km for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologs in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology.
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Mar 2023
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[16637]
Open Access
Abstract: The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the Enterobacteriaceae. ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of the Wzy membrane protein polymerase superfamily. WzyE activity is regulated by a membrane protein polysaccharide co-polymerase, WzzE. Förster resonance energy transfer experiments demonstrate that WzyE and WzzE from Pectobacterium atrosepticum form a complex in vivo, and immunoblotting and cryo-electron microscopy (cryo-EM) analysis confirm a defined stoichiometry of approximately eight WzzE to one WzyE. Low-resolution cryo-EM reconstructions of the complex, aided by an antibody recognizing the C-terminus of WzyE, reveals WzyE sits in the central membrane lumen formed by the octameric arrangement of the transmembrane helices of WzzE. The pairing of Wzy and Wzz is found in polymerization systems for other bacterial polymers, including lipopolysaccharide O-antigens and capsular polysaccharides. The data provide new structural insight into a conserved mechanism for regulating polysaccharide chain length in bacteria.
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Mar 2023
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein “Wzx-Wzy” system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.
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Jul 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Src homology 2 domain-containing inositol phosphate phosphatase 2 (SHIP2) is one of the 10 human inositol phosphate 5-phosphatases. One of its physiological functions is dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate, PtdIns(3,4,5)P3. It is therefore a therapeutic target for pathophysiologies dependent on PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Therapeutic interventions are limited by the dearth of crystallographic data describing ligand/inhibitor binding. An active site-directed fluorescent probe facilitated screening of compound libraries for SHIP2 ligands. With two additional orthogonal assays, several ligands including galloflavin were identified as low micromolar Ki inhibitors. One ligand, an oxo-linked ethylene-bridged dimer of benzene 1,2,4-trisphosphate, was shown to be an uncompetitive inhibitor that binds to a regulatory site on the catalytic domain. We posit that binding of ligands to this site restrains L4 loop motions that are key to interdomain communications that accompany high catalytic activity with phosphoinositide substrate. This site may, therefore, be a future druggable target for medicinal chemistry.
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Mar 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Abstract: Inositol pentakisphosphate 2-kinase catalyzes the phosphorylation of the axial 2-OH of myo-inositol 1,3,4,5,6-pentakisphosphate for de novo synthesis of myo-inositol hexakisphosphate. Disruption of inositol pentakisphosphate 2-kinase profoundly influences cellular processes; from nuclear mRNA export and phosphate homeostasis in yeast and plants, to establishment of left-right asymmetry in zebra fish. We elaborate an active site fluorescent probe that allows high throughput screening of Arabidopsis inositol pentakisphosphate 2-kinase. We show that the probe has a binding constant comparable to the Km values of inositol phosphate substrates of this enzyme, and can be used to prospect for novel substrates and inhibitors of inositol phosphate kinases. We identify several micromolar Ki inhibitors and validate this approach by solving the crystal structure of protein in complex with purpurogallin. We additionally solve structures of protein in complexes with epimeric higher inositol phosphates. This probe may find utility in characterization of a wide family of inositol phosphate kinases.
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Aug 2018
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[14406]
Abstract: Wzz is an integral inner membrane protein involved in regulating the length of lipopolysaccharide O-antigen glycans and essential for the virulence of many Gram-negative pathogens. In all Wzz homologs, the large periplasmic domain is proposed to be anchored by two transmembrane helices, but no information is available for the transmembrane and cytosolic domains. Here we have studied purified oligomeric Wzz complexes using cryoelectron microscopy and resolved the transmembrane regions within a semi-continuous detergent micelle. The transmembrane helices of each monomer display a right-handed super-helical twist, and do not interact with the neighboring transmembrane domains. Modeling, flexible fitting and multiscale simulation approaches were used to study the full-length complex and to provide explanations for the influence of the lipid bilayer on its oligomeric status. Based on structural and in silico observations, we propose a new mechanism for O-antigen chain-length regulation that invokes synergy of Wzz and its polymerase partner, Wzy.
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Apr 2017
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B23-Circular Dichroism
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[11051, 12036]
Open Access
Abstract: Long-chain bacterial polysaccharides have important roles in pathogenicity. In Escherichia coli O9a, a model for ABC transporter–dependent polysaccharide assembly, a large extracellular carbohydrate with a narrow size distribution is polymerized from monosaccharides by a complex of two proteins, WbdA (polymerase) and WbdD (terminating protein). Combining crystallography and small-angle X-ray scattering, we found that the C-terminal domain of WbdD contains an extended coiled-coil that physically separates WbdA from the catalytic domain of WbdD. The effects of insertions and deletions in the coiled-coil region were analyzed in vivo, revealing that polymer size is controlled by varying the length of the coiled-coil domain. Thus, the coiled-coil domain of WbdD functions as a molecular ruler that, along with WbdA:WbdD stoichiometry, controls the chain length of a model bacterial polysaccharide.polymerized from monosaccharides by a complex of two proteins, WbdA (polymerase) and WbdD (terminating protein).
Combining crystallography and small-angle X-ray scattering, we found that the C-terminal domain of WbdD contains an extended coiled-coil that physically separates WbdA from the catalytic domain of WbdD. The effects of insertions and deletions in the coiled-coil region were analyzed in vivo, revealing that polymer size is controlled by varying the length of the coiled-coil domain. Thus, the coiled-coil domain of WbdD functions as a molecular ruler that, along with WbdA:WbdD stoichiometry, controls the chain length of a model bacterial polysaccharide.
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Dec 2014
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I04-Macromolecular Crystallography
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Open Access
Abstract: WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations.
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Oct 2012
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