B21-High Throughput SAXS
I03-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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I04-1-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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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12579]
Open Access
Abstract: Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction with a yield of ~1-4 mg kg-1 of fruit. Due to toxicity, microbial production provides only low yields of up to 5-100 mg L-1. Herein, we report an efficient cell-free strategy to probe a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one (HBA), into raspberry ketone at up to 100% conversion. As part of this strategy, it is essential to recycle inexpensive cofactors. Specifically, the final enzyme step in the pathway is catalysed by raspberry ketone/zingerone synthase (RZS1), an NADPH-dependent double bond reductase. To relax cofactor specificity towards NADH, the preferred cofactor for cell-free biosynthesis, we identify a variant (G191D) with strong activity with NADH. We implement the RZS1 G191D variant within a ‘one-pot’ cell-free reaction to produce raspberry ketone at high-yield (61 mg L-1), which provides an alternative route to traditional microbial production. In conclusion, our cell-free strategy complements the growing interest in engineering synthetic enzyme cascades towards industrially relevant value-added chemicals.
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Aug 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23620]
Open Access
Abstract: UTP-glucose-1-phosphate uridylyltransferases (UGPases) are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UGPases, are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When wild-type and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen limited growth condition.
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Feb 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[16980]
Abstract: Several pathologies have been associated with the AAA + ATPase p97, an enzyme essential to protein homeostasis. Heterozygous polymorphisms in p97 have been shown to cause neurological disease, while elevated proteotoxic stress in tumours has made p97 an attractive cancer chemotherapy target. The cellular processes reliant on p97 are well described. High‐resolution structural models of its catalytic D2 domain, however, have proved elusive, as has the mechanism by which p97 converts the energy from ATP hydrolysis into mechanical force to unfold protein substrates. Here, we describe the high‐resolution structure of the p97 D2 ATPase domain. This crystal system constitutes a valuable tool for p97 inhibitor development and identifies a potentially druggable pocket in the D2 domain. In addition, its P61 symmetry suggests a mechanism for substrate unfolding by p97.
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Nov 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Thomas E.
Wood
,
Sophie A.
Howard
,
Andreas
Forster
,
Laura M.
Nolan
,
Eleni
Manoli
,
Nathan P.
Bullen
,
Hamish C. L.
Yau
,
Abderrahman
Hachani
,
Richard D.
Hayward
,
John C.
Whitney
,
Waldemar
Vollmer
,
Paul S.
Freemont
,
Alain
Filloux
Open Access
Abstract: The type VI secretion system (T6SS) is crucial in interbacterial competition and is a virulence determinant of many Gram-negative bacteria. Several T6SS effectors are covalently fused to secreted T6SS structural components such as the VgrG spike for delivery into target cells. In Pseudomonas aeruginosa, the VgrG2b effector was previously proposed to mediate bacterial internalization into eukaryotic cells. In this work, we find that the VgrG2b C-terminal domain (VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein. We resolve the structure of VgrG2bC-ter and confirm it is a member of the zinc-metallopeptidase family of enzymes. We show that this effector causes membrane blebbing at midcell, which suggests a distinct type of T6SS-mediated growth inhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Our study introduces a further effector family to the T6SS arsenal and demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.
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Oct 2019
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12579, 17221]
Open Access
Abstract: c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.
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Jan 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: NExo is an enzyme from Neisseria meningitidis that is specialized in the removal of the 3′-phosphate and other 3′-lesions, which are potential blocks for DNA repair. NExo is a highly active DNA 3′-phosphatase, and although it is from the class II AP family it lacks AP endonuclease activity. In contrast, the NExo homologue NApe, lacks 3′-phosphatase activity but is an efficient AP endonuclease. These enzymes act together to protect the meningococcus from DNA damage arising mainly from oxidative stress and spontaneous base loss. In this work, we present crystal structures of the specialized 3′-phosphatase NExo bound to DNA in the presence and absence of a 3′-phosphate lesion. We have outlined the reaction mechanism of NExo, and using point mutations we bring mechanistic insights into the specificity of the 3′-phosphatase activity of NExo. Our data provide further insight into the molecular origins of plasticity in substrate recognition for this class of enzymes. From this we hypothesize that these specialized enzymes lead to enhanced efficiency and accuracy of DNA repair and that this is important for the biological niche occupied by this bacterium.
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Oct 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13212]
Open Access
Abstract: Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction.
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Jan 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7299, 1227, 9424]
Open Access
Abstract: The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures, and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole-domain insertions by inserting a domain into one of the designed loops.
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Sep 2016
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