I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Open Access
Abstract: Cyclic di-GMP (c-di-GMP) is a second messenger that modulates multiple responses to environmental and cellular signals in bacteria. Here we identify CdbA, a DNA-binding protein of the ribbon-helix-helix family that binds c-di-GMP in Myxococcus xanthus. CdbA is essential for viability, and its depletion causes defects in chromosome organization and segregation leading to a block in cell division. The protein binds to the M. xanthus genome at multiple sites, with moderate sequence specificity; however, its depletion causes only modest changes in transcription. The interactions of CdbA with c-di-GMP and DNA appear to be mutually exclusive and residue substitutions in CdbA regions important for c-di-GMP binding abolish binding to both c-di-GMP and DNA, rendering these protein variants non-functional in vivo. We propose that CdbA acts as a nucleoid-associated protein that contributes to chromosome organization and is modulated by c-di-GMP, thus revealing a link between c-di-GMP signaling and chromosome biology.
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Apr 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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10369, 14692]
Open Access
Abstract: The bacterial second messenger cyclic-di-GMP is a widespread, prominent effector of lifestyle change. An example of this occurs in the predatory bacterium Bdellovibrio bacteriovorus, which cycles between free-living and intraperiplasmic phases after entering (and killing) another bacterium. The initiation of prey invasion is governed by DgcB (GGDEF enzyme) that produces cyclic-di-GMP in response to an unknown stimulus. Here, we report the structure of DgcB, and demonstrate that the GGDEF and sensory forkhead-associated (FHA) domains form an asymmetric dimer. Our structures indicate that the FHA domain is a consensus phosphopeptide sensor, and that the ligand for activation is surprisingly derived from the N-terminal region of DgcB itself. We confirm this hypothesis by determining the structure of a FHA:phosphopeptide complex, from which we design a constitutively-active mutant (confirmed via enzyme assays). Our results provide an understanding of the stimulus driving DgcB-mediated prey invasion and detail a unique mechanism of GGDEF enzyme regulation.
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Sep 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Rosalba
Lepore
,
Andriy
Kryshtafovych
,
Markus
Alahuhta
,
Harshul A.
Veraszto
,
Yannick J.
Bomble
,
Joshua C.
Bufton
,
Alex N.
Bullock
,
Cody
Caba
,
Hongnan
Cao
,
Owen R.
Davies
,
Ambroise
Desfosses
,
Matthew
Dunne
,
Krzysztof
Fidelis
,
Celia W.
Goulding
,
Manickam
Gurusaran
,
Irina
Gutsche
,
Christopher J.
Harding
,
Marcus D.
Hartmann
,
Christopher S.
Hayes
,
Andrzej
Joachimiak
,
Petr G.
Leiman
,
Peter
Loppnau
,
Andrew L.
Lovering
,
Vladimir V.
Lunin
,
Karolina
Michalska
,
Ignacio
Mir‐sanchis
,
Alok
Mitra
,
John
Moult
,
George N.
Phillips Jr
,
Daniel
Pinkas
,
Phoebe A.
Rice
,
Yufeng
Tong
,
Maya
Topf
,
Jonathan D.
Walton
,
Torsten
Schwede
Diamond Proposal Number(s):
[14692]
Open Access
Abstract: The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.
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Sep 2019
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I02-Macromolecular Crystallography
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Ian T.
Cadby
,
Sarah M
Basford
,
Ruth
Nottingham
,
Richard
Meek
,
Rebecca
Lowry
,
Carey
Lambert
,
Matthew
Tridgett
,
Rob
Till
,
Rashidah
Ahmad
,
Rowena
Fung
,
Laura
Hobley
,
William S.
Hughes
,
Patrick J.
Moynihan
,
R. Elizabeth
Sockett
,
Andrew L.
Lovering
Open Access
Abstract: Bacterial usage of the cyclic dinucleotide c‐di‐GMP is widespread, governing the transition between motile/sessile and unicellular/multicellular behaviors. There is limited information on c‐di‐GMP metabolism, particularly on regulatory mechanisms governing control of EAL c‐di‐GMP phosphodiesterases. Herein, we provide high‐resolution structures for an EAL enzyme Bd1971, from the predatory bacterium Bdellovibrio bacteriovorus, which is controlled by a second signaling nucleotide, cAMP. The full‐length cAMP‐bound form reveals the sensory N‐terminus to be a domain‐swapped variant of the cNMP/CRP family, which in the cAMP‐activated state holds the C‐terminal EAL enzyme in a phosphodiesterase‐active conformation. Using a truncation mutant, we trap both a half‐occupied and inactive apo‐form of the protein, demonstrating a series of conformational changes that alter juxtaposition of the sensory domains. We show that Bd1971 interacts with several GGDEF proteins (c‐di‐GMP producers), but mutants of Bd1971 do not share the discrete phenotypes of GGDEF mutants, instead having an elevated level of c‐di‐GMP, suggesting that the role of Bd1971 is to moderate these levels, allowing “action potentials” to be generated by each GGDEF protein to effect their specific functions.
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Jun 2019
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I04-Macromolecular Crystallography
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Gareth W.
Hughes
,
Stephen C. L.
Hall
,
Claire S.
Laxton
,
Pooja
Sridhar
,
Amirul H.
Mahadi
,
Caitlin
Hatton
,
Thomas J.
Piggot
,
Peter J.
Wotherspoon
,
Aneika C.
Leney
,
Douglas G.
Ward
,
Mohammed
Jamshad
,
Vaclav
Spana
,
Ian T.
Cadby
,
Christopher
Harding
,
Georgia L.
Isom
,
Jack A.
Bryant
,
Rebecca J.
Parr
,
Yasin
Yakub
,
Mark
Jeeves
,
Damon
Huber
,
Ian R.
Henderson
,
Luke A.
Clifton
,
Andrew L.
Lovering
,
Timothy J.
Knowles
Diamond Proposal Number(s):
[14692]
Abstract: The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA–OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.
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Jun 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: Growth and division by most bacteria requires remodelling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides, which are recycled in many model organisms. Bacteria and hosts can harness the unique nature of muropeptides as a signal for cell wall damage and infection, respectively. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core mycobacterial gene lpqI, encodes an authentic NagZ β-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.
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Jun 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6388]
Abstract: The peptidoglycan wall, located in the periplasm between the inner and outer membranes of the cell envelope in Gram-negative bacteria, maintains cell shape and endows osmotic robustness. Predatory Bdellovibrio bacteria invade the periplasm of other bacterial prey cells, usually crossing the peptidoglycan layer, forming transient structures called bdelloplasts within which the predators replicate. Prey peptidoglycan remains intact for several hours, but is modified and then degraded by escaping predators. Here we show predation is altered by deleting two Bdellovibrio N-acetylglucosamine (GlcNAc) deacetylases, one of which we show to have a unique two domain structure with a novel regulatory”plug”. Deleting the deacetylases limits peptidoglycan degradation and rounded prey cell “ghosts” persist after mutant-predator exit. Mutant predators can replicate unusually in the periplasmic region between the peptidoglycan wall and the outer membrane rather than between wall and inner-membrane, yet still obtain nutrients from the prey cytoplasm. Deleting two further genes encoding DacB/PBP4 family proteins, known to decrosslink and round prey peptidoglycan, results in a quadruple mutant Bdellovibrio which leaves prey-shaped ghosts upon predation. The resultant bacterial ghosts contain cytoplasmic membrane within bacteria-shaped peptidoglycan surrounded by outer membrane material which could have promise as “bacterial skeletons” for housing artificial chromosomes.
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May 2016
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Carey
Lambert
,
Ian T.
Cadby
,
Rob
Till
,
Nhat Khai
Bui
,
Thomas R.
Lerner
,
William S.
Hughes
,
David J.
Lee
,
Luke J.
Alderwick
,
Waldemar
Vollmer
,
Elizabeth R.
Sockett
,
Andrew L
Lovering
Diamond Proposal Number(s):
[8359, 10369]
Open Access
Abstract: Predatory Bdellovibrio bacteriovorus are natural antimicrobial organisms, killing other bacteria by whole-cell invasion. Self-protection against prey-metabolizing enzymes is important for the evolution of predation. Initial prey entry involves the predator's peptidoglycan DD-endopeptidases, which decrosslink cell walls and prevent wasteful entry by a second predator. Here we identify and characterize a self-protection protein from B. bacteriovorus, Bd3460, which displays an ankyrin-based fold common to intracellular pathogens of eukaryotes. Co-crystal structures reveal Bd3460 complexation of dual targets, binding a conserved epitope of each of the Bd3459 and Bd0816 endopeptidases. Complexation inhibits endopeptidase activity and cell wall decrosslinking in vitro. Self-protection is vital - ΔBd3460 Bdellovibrio deleteriously decrosslink self-peptidoglycan upon invasion, adopt a round morphology, and lose predatory capacity and cellular integrity. Our analysis provides the first mechanistic examination of self-protection in Bdellovibrio, documents protection-multiplicity for products of two different genomic loci, and reveals an important evolutionary adaptation to an invasive predatory bacterial lifestyle
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Dec 2015
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6388]
Open Access
Abstract: Bdellovibrio are predatory bacteria that have evolved to invade virtually all Gram-negative bacteria, including many prominent pathogens. Upon invasion, prey bacteria become rounded up into an osmotically stable niche for the Bdellovibrio, preventing further superinfection and allowing Bdellovibrio to replicate inside without competition, killing the prey bacterium and degrading its contents. Historically, prey rounding was hypothesized to be associated with peptidoglycan (PG) metabolism; we found two Bdellovibrio genes, bd0816 and bd3459, expressed at prey entry and encoding proteins with limited homologies to conventional dacB/PBP4 DD-endo/carboxypeptidases (responsible for peptidoglycan maintenance during growth and division). We tested possible links between Bd0816/3459 activity and predation. Bd3459, but not an active site serine mutant protein, bound ?-lactam, exhibited DD-endo/carboxypeptidase activity against purified peptidoglycan and, importantly, rounded up E. coli cells upon periplasmic expression. A ?Bd0816 ?Bd3459 double mutant invaded prey more slowly than the wild type (with negligible prey cell rounding) and double invasions of single prey by more than one Bdellovibrio became more frequent. We solved the crystal structure of Bd3459 to 1.45 Å and this revealed predation-associated domain differences to conventional PBP4 housekeeping enzymes (loss of the regulatory domain III, alteration of domain II and a more exposed active site). The Bd3459 active site (and by similarity the Bd0816 active site) can thus accommodate and remodel the various bacterial PGs that Bdellovibrio may encounter across its diverse prey range, compared to the more closed active site that “regular” PBP4s have for self cell wall maintenance. Therefore, during evolution, Bdellovibrio peptidoglycan endopeptidases have adapted into secreted predation-specific proteins, preventing wasteful double invasion, and allowing activity upon the diverse prey peptidoglycan structures to sculpt the prey cell into a stable intracellular niche for replication.
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Feb 2012
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