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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18145]
Open Access
Abstract: Primary hyperoxaluria type I (PH1) is caused by AGXT gene mutations that decrease the functional activity of alanine:glyoxylate aminotransferase. A build-up of the enzyme’s substrate, glyoxylate, results in excessive deposition of calcium oxalate crystals in the renal tract, leading to debilitating renal failure. Oxidation of glycolate by glycolate oxidase (or hydroxy acid oxidase 1, HAO1) is a major cellular source of glyoxylate, and siRNA studies have shown phenotypic rescue of PH1 by the knockdown of HAO1, representing a promising inhibitor target. Here, we report the discovery and optimization of six low-molecular-weight fragments, identified by crystallography-based fragment screening, that bind to two different sites on the HAO1 structure: at the active site and an allosteric pocket above the active site. The active site fragments expand known scaffolds for substrate-mimetic inhibitors to include more chemically attractive molecules. The allosteric fragments represent the first report of non-orthosteric inhibition of any hydroxy acid oxidase and hold significant promise for improving inhibitor selectivity. The fragment hits were verified to bind and inhibit HAO1 in solution by fluorescence-based activity assay and surface plasmon resonance. Further optimization cycle by crystallography and biophysical assays have generated two hit compounds of micromolar (44 and 158 µM) potency that do not compete with the substrate and provide attractive starting points for the development of potent and selective HAO1 inhibitors.
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May 2022
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Krios I-Titan Krios I at Diamond
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John J.
Kelly
,
Dale
Tranter
,
Els
Pardon
,
Gamma
Chi
,
Holger
Kramer
,
Lotta
Happonen
,
Kelly M.
Knee
,
Jay M.
Janz
,
Jan
Steyaert
,
Christine
Bulawa
,
Ville O.
Paavilainen
,
Juha T.
Huiskonen
,
Wyatt W.
Yue
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.
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Apr 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[20221]
Open Access
Abstract: Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.
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Oct 2021
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I03-Macromolecular Crystallography
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Igor M.
Ferreira
,
José Edwin N.
Quesñay
,
Alliny C. S.
Bastos
,
Camila T.
Rodrigues
,
Melanie
Vollmar
,
Tobias
Krojer
,
Claire
Strain-Damerell
,
Nicola A.
Burgess-Brown
,
Frank
Von Delft
,
Wyatt W.
Yue
,
Sandra M G.
Dias
,
Andre L. B.
Ambrosio
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called “lid loop” is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS “activation” loop, formerly known as the “gating” loop) renders a highly active protein in stable tetrameric form. We conclude that the “activation” loop, a known target for GLS inhibition, may also be a drug target for GLS2.
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Jun 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Sabrina R.
Mackinnon
,
Tobias
Krojer
,
William R.
Foster
,
Laura
Diaz-Saez
,
Manshu
Tang
,
Kilian V. M.
Huber
,
Frank
Von Delft
,
Kent
Lai
,
Paul
Brennan
,
Gustavo
Arruda Bezerra
,
Wyatt W.
Yue
Diamond Proposal Number(s):
[18145]
Abstract: Classic galactosemia is caused by loss-of-function mutations in
galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic
accumulation of its substrate, galactose-1-phosphate. One proposed therapy
is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by
galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1)
are primarily ATP-competitive with limited clinical utility to date. Here, we
determined crystal structures of hGALK1 bound with reported ATP-
competitive inhibitors of the spiro-benzoxazole series, to reveal their binding
mode in the active site. Spurred by the need for additional chemotypes of
hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also
performed crystallography-based screening by soaking hundreds of hGALK1
crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.
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Mar 2021
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Gustavo A.
Bezerra
,
William R.
Foster
,
Henry J.
Bailey
,
Kevin G.
Hicks
,
Sven W.
Sauer
,
Bianca
Dimitrov
,
Thomas J.
Mccorvie
,
Jürgen G.
Okun
,
Jared
Rutter
,
Stefan
Kölker
,
Wyatt W.
Yue
Open Access
Abstract: DHTKD1 is a lesser-studied E1 enzyme among the family of 2-oxoacid dehydrogenases. In complex with E2 (dihydrolipoamide succinyltransferase, DLST) and E3 (dihydrolipoamide dehydrogenase, DLD) components, DHTKD1 is involved in lysine and tryptophan catabolism by catalysing the oxidative decarboxylation of 2-oxoadipate (2OA) in mitochondria. Here, the 1.9 Å resolution crystal structure of human DHTKD1 is solved in complex with the thiamine diphosphate co-factor. The structure reveals how the DHTKD1 active site is modelled upon the well characterized homologue 2-oxoglutarate (2OG) dehydrogenase but engineered specifically to accommodate its preference for the longer substrate of 2OA over 2OG. A 4.7 Å resolution reconstruction of the human DLST catalytic core is also generated by single-particle electron microscopy, revealing a 24-mer cubic scaffold for assembling DHTKD1 and DLD protomers into a megacomplex. It is further demonstrated that missense DHTKD1 variants causing the inborn error of 2-aminoadipic and 2-oxoadipic aciduria impact on the complex formation, either directly by disrupting the interaction with DLST, or indirectly through destabilizing the DHTKD1 protein. This study provides the starting framework for developing DHTKD1 modulators to probe the intricate mitochondrial energy metabolism.
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Jul 2020
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Henry J.
Bailey
,
Gustavo A.
Bezerra
,
Jason R.
Marcero
,
Siladitya
Padhi
,
William R.
Foster
,
Elzbieta
Rembeza
,
Arijit
Roy
,
David F.
Bishop
,
Robert J.
Desnick
,
Gopalakrishnan
Bulusu
,
Harry A.
Dailey
,
Wyatt W.
Yue
Open Access
Abstract: 5′-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.
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Jun 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21035, 10619]
Open Access
Abstract: The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich’s ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein–protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.
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May 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Giancarlo
Abis
,
Rebecca L.
Charles
,
Jolanta
Kopec
,
Wyatt W.
Yue
,
R. Andrew
Atkinson
,
Tam T. T.
Bui
,
Steven
Lynham
,
Simona
Popova
,
Yin-Biao
Sun
,
Franca
Fraternali
,
Philip
Eaton
,
Maria R.
Conte
Diamond Proposal Number(s):
[13597, 10619]
Open Access
Abstract: Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.
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May 2019
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