I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[13775]
Abstract: The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5′-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.
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Mar 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Abstract: Cycloaddition reactions generate chemical complexity in a single step. Here we report the crystal structures of three homologous plant-derived cyclases involved in the biosynthesis of iboga and aspidosperma alkaloids. These enzymes act on the same substrate, named angryline, to generate three distinct scaffolds. Mutational analysis reveals how these highly similar enzymes control regio- and stereo-selectivity.
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Feb 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043]
Abstract: The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.
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Jan 2020
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I24-Microfocus Macromolecular Crystallography
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Matilde
De Las Rivas
,
Earnest James
Paul Daniel
,
Yoshiki
Narimatsu
,
Ismael
Compañón
,
Kentaro
Kato
,
Pablo
Hermosilla
,
Aurélien
Thureau
,
Laura
Ceballos-laita
,
Helena
Coelho
,
Pau
Bernadó
,
Filipa
Marcelo
,
Lars
Hansen
,
Ryota
Maeda
,
Anabel
Lostao
,
Francisco
Corzana
,
Henrik
Clausen
,
Thomas A.
Gerken
,
Ramon
Hurtado-guerrero
Diamond Proposal Number(s):
[14739]
Abstract: Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3’s structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.
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Jan 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Abstract: Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.
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Nov 2019
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I23-Long wavelength MX
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Amalie F.
Rudolf
,
Maia
Kinnebrew
,
Christiane
Kowatsch
,
T. Bertie
Ansell
,
Kamel
El Omari
,
Benjamin
Bishop
,
Els
Pardon
,
Rebekka A.
Schwab
,
Tomas
Malinauskas
,
Mingxing
Qian
,
Ramona
Duman
,
Douglas F.
Covey
,
Jan
Steyaert
,
Armin
Wagner
,
Mark S. P.
Sansom
,
Rajat
Rohatgi
,
Christian
Siebold
Abstract: Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids—a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.
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Oct 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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William
Farnaby
,
Manfred
Koegl
,
Michael J.
Roy
,
Claire
Whitworth
,
Emelyne
Diers
,
Nicole
Trainor
,
David
Zollman
,
Steffen
Steurer
,
Jale
Karolyi-oezguer
,
Carina
Riedmueller
,
Teresa
Gmaschitz
,
Johannes
Wachter
,
Christian
Dank
,
Michael
Galant
,
Bernadette
Sharps
,
Klaus
Rumpel
,
Elisabeth
Traxler
,
Thomas
Gerstberger
,
Renate
Schnitzer
,
Oliver
Petermann
,
Peter
Greb
,
Harald
Weinstabl
,
Gerd
Bader
,
Andreas
Zoephel
,
Alexander
Weiss-puxbaum
,
Katharina
Ehrenhöfer-wölfer
,
Simon
Wöhrle
,
Guido
Boehmelt
,
Joerg
Rinnenthal
,
Heribert
Arnhof
,
Nicola
Wiechens
,
Meng-ying
Wu
,
Tom
Owen-hughes
,
Peter
Ettmayer
,
Mark
Pearson
,
Darryl B.
Mcconnell
,
Alessio
Ciulli
Diamond Proposal Number(s):
[14980]
Abstract: Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.
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Jun 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9426]
Abstract: GCN5-related N-acetyl-transferase (GNAT)-like enzymes from toxin–antitoxin modules are strong inhibitors of protein synthesis. Here, we present the bases of the regulatory mechanisms of ataRT, a model GNAT-toxin–antitoxin module, from toxin synthesis to its action as a transcriptional de-repressor. We show the antitoxin (AtaR) traps the toxin (AtaT) in a pre-catalytic monomeric state and precludes the effective binding of ac-CoA and its target Met-transfer RNAfMet. In the repressor complex, AtaR intrinsically disordered region interacts with AtaT at two different sites, folding into different structures, that are involved in two separate functional roles, toxin neutralization and placing the DNA-binding domains of AtaR in a binding-compatible orientation. Our data suggests AtaR neutralizes AtaT as a monomer, right after its synthesis and only the toxin–antitoxin complex formed in this way is an active repressor. Once activated by dimerization, later neutralization of the toxin results in a toxin–antitoxin complex that is not able to repress transcription.
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Feb 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Abstract: Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effects in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD+ yet does not utilize it chemically for a non-oxidoreductive formal [4 + 2] cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.
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Dec 2018
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I04-Macromolecular Crystallography
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Suzana
Markolovic
,
Qinqin
Zhuang
,
Sarah E.
Wilkins
,
Charlotte D.
Eaton
,
Martine I.
Abboud
,
Maximiliano J.
Katz
,
Helen E.
Mcneil
,
Robert K.
Leśniak
,
Charlotte
Hall
,
Weston B.
Struwe
,
Rebecca
Konietzny
,
Simon
Davis
,
Ming
Yang
,
Wei
Ge
,
Justin L. P.
Benesch
,
Benedikt M.
Kessler
,
Peter J.
Ratcliffe
,
Matthew E.
Cockman
,
Roman
Fischer
,
Pablo
Wappner
,
Rasheduzzaman
Chowdhury
,
Mathew L.
Coleman
,
Christopher J.
Schofield
Abstract: Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(ii)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.
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Jun 2018
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