I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[31800]
Open Access
Abstract: Two major glycosaminoglycan types, heparan sulfate (HS) and chondroitin sulfate (CS), control many aspects of development and physiology in a type-specific manner. HS and CS are attached to core proteins via a common linker tetrasaccharide, but differ in their polymer backbones. How core proteins are specifically modified with HS or CS has been an enduring mystery. By reconstituting glycosaminoglycan biosynthesis in vitro, we establish that the CS-initiating N-acetylgalactosaminyltransferase CSGALNACT2 modifies all glycopeptide substrates equally, whereas the HS-initiating N-acetylglucosaminyltransferase EXTL3 is selective. Structure-function analysis reveals that acidic residues in the glycopeptide substrate and a basic exosite in EXTL3 are critical for specifying HS biosynthesis. Linker phosphorylation by the xylose kinase FAM20B accelerates linker synthesis and initiation of both HS and CS, but has no effect on the subsequent polymerisation of the backbone. Our results demonstrate that modification with CS occurs by default and must be overridden by EXTL3 to produce HS.
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Oct 2023
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I03-Macromolecular Crystallography
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Christopher D. M.
Hutchison
,
James
Baxter
,
Ann
Fitzpatrick
,
Gabriel
Dorlhiac
,
Alisia
Fadini
,
Samuel
Perrett
,
Karim
Maghlaoui
,
Salomé
Bodet Lefèvre
,
Violeta
Cordon-Preciado
,
Josie L.
Ferreira
,
Volha U.
Chukhutsina
,
Douglas
Garratt
,
Jonathan
Barnard
,
Gediminas
Galinis
,
Flo
Glencross
,
Rhodri M.
Morgan
,
Sian
Stockton
,
Ben
Taylor
,
Letong
Yuan
,
Matthew G.
Romei
,
Chi-Yun
Lin
,
Jon P.
Marangos
,
Marius
Schmidt
,
Viktoria
Chatrchyan
,
Tiago
Buckup
,
Dmitry
Morozov
,
Jaehyun
Park
,
Sehan
Park
,
Intae
Eom
,
Minseok
Kim
,
Dogeun
Jang
,
Hyeongi
Choi
,
Hyojung
Hyun
,
Gisu
Park
,
Eriko
Nango
,
Rie
Tanaka
,
Shigeki
Owada
,
Kensuke
Tono
,
Daniel P.
Deponte
,
Sergio
Carbajo
,
Matt
Seaberg
,
Andrew
Aquila
,
Sebastien
Boutet
,
Anton
Barty
,
So
Iwata
,
Steven G.
Boxer
,
Gerrit
Groenhof
,
Jasper J.
Van Thor
Diamond Proposal Number(s):
[22819, 17221]
Open Access
Abstract: The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump–probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.
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Aug 2023
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I23-Long wavelength MX
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Alisia
Fadini
,
Christopher D. M.
Hutchison
,
Dmitry
Morozov
,
Jeffrey
Chang
,
Karim
Maghlaoui
,
Samuel
Perrett
,
Fangjia
Luo
,
Jeslyn C. X.
Kho
,
Matthew G.
Romei
,
R. Marc L.
Morgan
,
Christian
Orr
,
Violeta
Cordon-Preciado
,
Takaaki
Fujiwara
,
Nipawan
Nuemket
,
Takehiko
Tosha
,
Rie
Tanaka
,
Shigeki
Owada
,
Kensuke
Tono
,
So
Iwata
,
Steven G.
Boxer
,
Gerrit
Groenhof
,
Eriko
Nango
,
Jasper J.
Van Thor
Diamond Proposal Number(s):
[23620]
Open Access
Abstract: Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements.
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Jul 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23620]
Open Access
Abstract: Background: Hyaluronic acid (HA) is a major polysaccharide component of the extracellular matrix. HA has essential functions in tissue architecture and the regulation of cell behaviour. HA turnover needs to be finely balanced. Increased HA degradation is associated with cancer, inflammation, and other pathological situations. Transmembrane protein 2 (TMEM2) is a cell surface protein that has been reported to degrade HA into ~5 kDa fragments and play an essential role in systemic HA turnover.
Methods: We produced the soluble TMEM2 ectodomain (residues 106-1383; sTMEM2) in human embryonic kidney cells (HEK293) and determined its structure using X-ray crystallography. We tested sTMEM2 hyaluronidase activity using fluorescently labelled HA and size fractionation of reaction products. We tested HA binding in solution and using a glycan microarray.
Results: Our crystal structure of sTMEM2 confirms a remarkably accurate prediction by AlphaFold. sTMEM2 contains a parallel β-helix typical of other polysaccharide-degrading enzymes, but an active site cannot be assigned with confidence. A lectin-like domain is inserted into the β-helix and predicted to be functional in carbohydrate binding. A second lectin-like domain at the C-terminus is unlikely to bind carbohydrates. We did not observe HA binding in two assay formats, suggesting a modest affinity at best. Unexpectedly, we were unable to observe any HA degradation by sTMEM2. Our negative results set an upper limit for kcat of approximately 10-5 min-1.
Conclusions: Although sTMEM2 contains domain types consistent with its suggested role in TMEM2 degradation, its hyaluronidase activity was undetectable. HA degradation by TMEM2 may require additional proteins and/or localisation at the cell surface.
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May 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23620]
Open Access
Abstract: Background: Hyaluronic acid (HA) is a major polysaccharide component of the extracellular matrix. HA has essential functions in tissue architecture and the regulation of cell behaviour. HA turnover needs to be finely balanced. Increased HA degradation is associated with cancer, inflammation, and other pathological situations. Transmembrane protein 2 (TMEM2) is a cell surface protein that has been reported to degrade HA into ~5 kDa fragments and play an essential role in systemic HA turnover.
Methods: We produced the soluble TMEM2 ectodomain (residues 106-1383; sTMEM2) in human embryonic kidney cells (HEK293) and determined its structure using X-ray crystallography. We tested sTMEM2 hyaluronidase activity using fluorescently labelled HA and size fractionation of reaction products.
Results: Our crystal structure of sTMEM2 confirms a remarkably accurate prediction by AlphaFold. sTMEM2 contains a parallel β-helix typical of other polysaccharide-degrading enzymes, but an active site cannot be assigned with confidence. A lectin-like domain is inserted into the β-helix and predicted to be functional in carbohydrate binding. A second lectin-like domain at the C-terminus is unlikely to bind carbohydrates. Unexpectedly, we were unable to observe any HA degradation by sTMEM2. Our negative results set an upper limit for kcat of approximately 10-5 min-1.
Conclusions: Although sTMEM2 contains domain types consistent with its suggested role in TMEM2 degradation, its hyaluronidase activity was undetectable. HA degradation by TMEM2 may require additional proteins and/or localisation at the cell surface.
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Feb 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I23-Long wavelength MX
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Diamond Proposal Number(s):
[17221]
Open Access
Abstract: Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows concerted isomerization of C9’-C8’ and C7’-C6’ single bonds in the bicycle-pedal (s-BP) manner and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven recovery of the s-BP isomer to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. Solution FTIR and UV/Vis spectroscopy support the single bond isomerization of the carotenoid in the s-BP manner and subsequent thermal structural reactions as the basis of OCP photoreception.
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Oct 2022
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I02-Macromolecular Crystallography
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Mostafa
Jamshidiha
,
Thomas
Lanyon-Hogg
,
Charlotte L.
Sutherell
,
Gregory B.
Craven
,
Montse
Tersa
,
Elena
De Vita
,
Delia
Brustur
,
Inmaculada
Perez-Dorado
,
Sarah
Hassan
,
Rita
Petracca
,
Rhodri M.
Morgan
,
Máximo
Sanz-Hernández
,
Jim C.
Norman
,
Alan
Armstrong
,
David J.
Mann
,
Ernesto
Cota
,
Edward W.
Tate
Diamond Proposal Number(s):
[17221, 23620]
Open Access
Abstract: Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein–protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket (‘WF-binding pocket’) via a conserved tryptophan–phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.
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Dec 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12579]
Open Access
Abstract: Natural products and their analogues are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds, which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogues of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via the Suzuki–Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein we demonstrate a flexible and rapid approach to access a diverse chemical space that can be applied to a wide range of natural product scaffolds.
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Oct 2021
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Open Access
Abstract: Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers, and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.
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Oct 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17221]
Abstract: Toxin–antitoxin (TA) systems are a large family of genes implicated in the regulation of bacterial growth and its arrest in response to attacks. These systems encode nonsecreted toxins and antitoxins that specifically pair, even when present in several paralogous copies per genome. Salmonella enterica serovar Typhimurium contains three paralogous TacAT systems that block bacterial translation. We determined the crystal structures of the three TacAT complexes to understand the structural basis of specific TA neutralization and the evolution of such specific pairing. In the present study, we show that alteration of a discrete structural add-on element on the toxin drives specific recognition by their cognate antitoxin underpinning insulation of the three pairs. Similar to other TA families, the region supporting TA-specific pairing is key to neutralization. Our work reveals that additional TA interfaces beside the main neutralization interface increase the safe space for evolution of pairing specificity.
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Sep 2021
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