Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Glycogen synthase (GYS1) is the central enzyme in muscle glycogen biosynthesis. GYS1 activity is inhibited by phosphorylation of its amino (N) and carboxyl (C) termini, which is relieved by allosteric activation of glucose-6-phosphate (Glc6P). We present cryo-EM structures at 3.0–4.0 Å resolution of phosphorylated human GYS1, in complex with a minimal interacting region of glycogenin, in the inhibited, activated and catalytically competent states. Phosphorylations of specific terminal residues are sensed by different arginine clusters, locking the GYS1 tetramer in an inhibited state via intersubunit interactions. The Glc6P activator promotes conformational change by disrupting these interactions and increases the flexibility of GYS1, such that it is poised to adopt a catalytically competent state when the sugar donor UDP-glucose (UDP-glc) binds. We also identify an inhibited-like conformation that has not transitioned into the activated state, in which the locking interaction of phosphorylation with the arginine cluster impedes subsequent conformational changes due to Glc6P binding. Our results address longstanding questions regarding the mechanism of human GYS1 regulation.
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Jul 2022
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I04-Macromolecular Crystallography
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Kathrin
Heuberger
,
Henry J.
Bailey
,
Patricie
Burda
,
Apirat
Chaikuad
,
Ewelina
Krysztofinska
,
Terttu
Suormala
,
Céline
Bürer
,
Seraina
Lutz
,
Brian
Fowler
,
D. Sean
Froese
,
Wyatt W.
Yue
,
Matthias R.
Baumgartner
Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of d-methylmalonyl-CoA and l-methylmalonyl-CoA in propionate catabolism. Autosomal recessive pathogenic variations in MCEE reportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with pathogenic variations in MCEE. Nine patients were homozygous for the known nonsense variation p.Arg47* (c.139C > T), and one for the novel missense variation p.Ile53Arg (c.158T > G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described pathogenic variations p.Lys60Gln and p.Arg143Cys, onto our 1.8 Å structure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. We solved the structure of MCEE-Arg143Cys to 1.9 Å and found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this variation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.
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Jan 2019
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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D. Sean
Froese
,
Jolanta
Kopec
,
Elzbieta
Rembeza
,
Gustavo
Arruda Bezerra
,
Anselm Erich
Oberholzer
,
Terttu
Suormala
,
Seraina
Lutz
,
Rod
Chalk
,
Oktawia
Borkowska
,
Matthias R.
Baumgartner
,
Wyatt W.
Yue
Diamond Proposal Number(s):
[10619, 51433]
Open Access
Abstract: The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.
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Jun 2018
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I03-Macromolecular Crystallography
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Moniek
Riemersma
,
Sean
Froese
,
Walinka
Van tol
,
Udo f
Engelke
,
Jolanta
Kopec
,
Monique
Van scherpenzeel
,
Angel
Ashikov
,
Tobias
Krojer
,
Frank
Von Delft
,
Marco
Tessari
,
Anna
Buczkowska
,
Ewa
Swiezewska
,
Lucas t
Jae
,
Thijn r
Brummelkamp
,
Hiroshi
Manya
,
Tamao
Endo
,
Hans
Van bokhoven
,
Wyatt
Yue
,
Dirk j
Lefeber
Abstract: A unique, unsolved O-mannosyl glycan on alpha-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional alpha-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies.
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Dec 2015
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Sean
Froese
,
Jolanta
Kopec
,
Fiona
Fitzpatrick
,
Marion
Schuller
,
Thomas
Mccorvie
,
Rod
Chalk
,
Tanja
Plessl
,
Victoria
Fettelschoss
,
Brian
Fowler
,
Matthias R.
Baumgartner
,
Wyatt
Yue
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Background: Two intracellular proteins,
MMACHC and MMADHC, functionally interact
for cobalamin trafficking.
Results: MMADHC crystal structure reveals
protein-interacting regions and unexpected
homology to MMACHC; mutations on either
protein interfere with complex formation via
different mechanisms.
Conclusion: Complex formation likely depends on
prior cobalamin processing and can be broken by
disease mutations.
Significance: MMACHC-MMADHC
heterodimerization forms the essential trafficking
chaperone delivering cobalamin to client enzymes.
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Oct 2015
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Sean
Froese
,
Amit
Michaeli
,
Thomas
Mccorvie
,
Tobias
Krojer
,
Meitav
Sasi
,
Esther
Melaev
,
Amiram
Goldblum
,
Maria
Zatsepin
,
Alexander
Lossos
,
Rafael
Álvarez
,
Pablo V.
Escribá
,
Berge A.
Minassian
,
Frank
Von Delft
,
Or
Kakhlon
,
Wyatt
Yue
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Glycogen branching enzyme 1 (GBE1) plays an essential role in glycogen biosynthesis by generating α-1,6-glucosidic branches from α-1,4-linked glucose chains, to increase solubility of the glycogen polymer. Mutations in the GBE1 gene lead to the heterogeneous early-onset glycogen storage disorder type IV (GSDIV) or the late-onset adult polyglucosan body disease (APBD). To better understand this essential enzyme, we crystallized human GBE1 in the apo form, and in complex with a tetra- or hepta-saccharide. The GBE1 structure reveals a conserved amylase core that houses the active centre for the branching reaction and harbours almost all GSDIV and APBD mutations. A non-catalytic binding cleft, proximal to the site of the common APBD mutation p.Y329S, was found to bind the tetra- and hepta-saccharides and may represent a higher-affinity site employed to anchor the complex glycogen substrate for the branching reaction. Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization. To explore this, we generated a structural model of GBE1-p.Y329S and designed peptides ab initio to stabilize the mutation. As proof-of-principle, we evaluated treatment of one tetra-peptide, Leu-Thr-Lys-Glu, in APBD patient cells. We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells. Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.
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Sep 2015
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I03-Macromolecular Crystallography
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Open Access
Abstract: In humans, the gene encoding a reverse
thymidylate synthase (rTS) is transcribed in the reverse
direction of the gene encoding thymidylate synthase (TS) that
is involved in DNA biosynthesis. Three isoforms are found: á,
â, and ã, with the transcript of the á-isoform overlapping with
that of TS. rTSâ has been of interest since the discovery of its
overexpression in methotrexate and 5-fluorouracil resistant cell
lines. Despite more than 20 years of study, none of the rTS
isoforms have been biochemically or structurally characterized.
In this study, we identified rTSã as an L-fuconate dehydratase
and determined its high-resolution crystal structure. Our data provide an explanation for the observed difference in enzymatic
activities between rTSâ and rTSã, enabling more informed proposals for the possible function of rTSâ in chemotherapeutic
resistance.
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Apr 2014
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Sean
Froese
,
Farhad
Forouhar
,
Timothy h.
Tran
,
Melanie
Vollmar
,
Yi seul
Kim
,
Scott
Lew
,
Helen
Neely
,
Jayaraman
Seetharaman
,
Yang
Shen
,
Rong
Xiao
,
Thomas b.
Acton
,
John K.
Everett
,
Giuseppe
Cannone
,
Sriharsha
Puranik
,
Pavel
Savitsky
,
Tobias
Krojer
,
Ewa
Pilka
,
Wasim
Kiyani
,
Wen hwa
Lee
,
Brian d.
Marsden
,
Frank
Von Delft
,
Charles K.
Allerston
,
Laura
Spagnolo
,
Opher
Gileadi
,
Gaetano T.
Montelione
,
Udo
Oppermann
,
Wyatt W.
Yue
,
Liang
Tong
Open Access
Abstract: Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.
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Jun 2013
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I24-Microfocus Macromolecular Crystallography
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Abstract: Defects in the MMACHC gene represent the most common disorder of cobalamin (Cbl) metabolism, affecting synthesis of the enzyme cofactors adenosyl-Cbl and methyl-Cbl. The encoded MMACHC protein binds intracellular Cbl derivatives with different upper axial ligands and exhibits flavin mononucleotide (FMN)-dependent decyanase activity toward cyano-Cbl as well as glutathione (GSH)-dependent dealkylase activity toward alkyl-Cbls. We determined the structure of human MMACHC·adenosyl-Cbl complex, revealing a tailor-made nitroreductase scaffold which binds adenosyl-Cbl in a “base-off, five-coordinate” configuration for catalysis. We further identified an arginine-rich pocket close to the Cbl binding site responsible for GSH binding and dealkylation activity. Mutation of these highly conserved arginines, including a replication of the prevalent MMACHC missense mutation, Arg161Gln, disrupts GSH binding and dealkylation. We further showed that two Cbl-binding monomers dimerize to mediate the reciprocal exchange of a conserved “PNRRP” loop from both subunits, serving as a protein cap for the upper axial ligand in trans and required for proper dealkylation activity. Our dimeric structure is supported by solution studies, where dimerization is triggered upon binding its substrate adenosyl-Cbl or cofactor FMN. Together our data provide a structural framework to understanding catalytic function and disease mechanism for this multifunctional enzyme.
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May 2012
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Abstract: Glycogenin initiates the synthesis of a maltosaccharide chain covalently attached to itself on Tyr195 via a stepwise glucosylation reaction, priming glycogen synthesis. We have captured crystallographic snapshots of human glycogenin during its reaction cycle, revealing a dynamic conformational switch between ground and active states mediated by the sugar donor UDP-glucose. This switch includes the ordering of a polypeptide stretch containing Tyr195, and major movement of an approximately 30-residue “lid” segment covering the active site. The rearranged lid guides the nascent maltosaccharide chain into the active site in either an intra- or intersubunit mode dependent upon chain length and steric factors and positions the donor and acceptor sugar groups for catalysis. The Thr83Met mutation, which causes glycogen storage disease XV, is conformationally locked in the ground state and catalytically inactive. Our data highlight the conformational plasticity of glycogenin and coexistence of two modes of glucosylation as integral to its catalytic mechanism.
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Dec 2011
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