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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Radoslaw P.
Nowak
,
Stephen L.
Deangelo
,
Dennis
Buckley
,
Zhixiang
He
,
Katherine A.
Donovan
,
Jian
An
,
Nozhat
Safaee
,
Mark P.
Jedrychowski
,
Charles M.
Ponthier
,
Mette
Ishoey
,
Tinghu
Zhang
,
Joseph D.
Mancias
,
Nathanael S.
Gray
,
James E.
Bradner
,
Eric S.
Fischer
Abstract: Heterobifunctional small-molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize a comprehensive characterization of the ligand-dependent CRBN–BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low-energy binding conformations that are selectively bound by ligands. We demonstrate that computational protein–protein docking can reveal the underlying interprotein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic interprotein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.
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Jun 2018
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12788]
Abstract: Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation–PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP–reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.
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Jul 2017
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Abstract: SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a β-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π–sugar interaction and reveal a quantum mechanics–molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.
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Jun 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Liang
Wu
,
Jianbing
Jiang
,
Yi
Jin
,
Wouter W.
Kallemeijn
,
Chi-lin
Kuo
,
Marta
Artola
,
Wei
Dai
,
Cas
Van Elk
,
Marco
Van Eijk
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codee
,
Bogdan I.
Florea
,
Johannes M. F. G.
Aerts
,
Herman S.
Overkleeft
,
Gideon J.
Davies
Diamond Proposal Number(s):
[9948, 13587]
Abstract: Humans express at least two distinct β-glucuronidase enzymes that are involved in disease: exo-acting β-glucuronidase (GUSB), whose deficiency gives rise to mucopolysaccharidosis type VII, and endo-acting heparanase (HPSE), whose overexpression is implicated in inflammation and cancers. The medical importance of these enzymes necessitates reliable methods to assay their activities in tissues. Herein, we present a set of β-glucuronidase-specific activity-based probes (ABPs) that allow rapid and quantitative visualization of GUSB and HPSE in biological samples, providing a powerful tool for dissecting their activities in normal and disease states. Unexpectedly, we find that the supposedly inactive HPSE proenzyme proHPSE is also labeled by our ABPs, leading to surprising insights regarding structural relationships between proHPSE, mature HPSE, and their bacterial homologs. Our results demonstrate the application of β-glucuronidase ABPs in tracking pathologically relevant enzymes and provide a case study of how ABP-driven approaches can lead to discovery of unanticipated structural and biochemical functionality.
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Jun 2017
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