I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17221]
Open Access
Abstract: Carbapenem-resistance in Klebsiella pneumoniae (KP) sequence type ST258 is mediated by carbapenemases (e.g. KPC-2) and loss or modification of the major non-selective porins OmpK35 and OmpK36. However, the mechanism underpinning OmpK36-mediated resistance and consequences of these changes on pathogenicity remain unknown. By solving the crystal structure of a clinical ST258 OmpK36 variant we provide direct structural evidence of pore constriction, mediated by a di-amino acid (Gly115-Asp116) insertion into loop 3, restricting diffusion of both nutrients (e.g. lactose) and Carbapenems. In the presence of KPC-2 this results in a 16-fold increase in MIC to Meropenem. Additionally, the Gly-Asp insertion impairs bacterial growth in lactose-containing medium and confers a significant in vivo fitness cost in a murine model of ventilator-associated pneumonia. Our data suggests that the continuous selective pressure imposed by widespread Carbapenem utilisation in hospital settings drives the expansion of KP expressing Gly-Asp insertion mutants, despite an associated fitness cost.
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Sep 2019
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I24-Microfocus Macromolecular Crystallography
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Simon R.
Bushell
,
Ashley C. W.
Pike
,
Maria E.
Falzone
,
Nils J. G.
Rorsman
,
Chau M.
Ta
,
Robin A.
Corey
,
Thomas D.
Newport
,
John C.
Christianson
,
Lara F.
Scofano
,
Chitra
Shintre
,
Annamaria
Tessitore
,
Amy
Chu
,
Qinrui
Wang
,
Leela
Shrestha
,
Shubhashish M. M.
Mukhopadhyay
,
James D.
Love
,
Nicola A.
Burgess-brown
,
Rebecca
Sitsapesan
,
Phillip J.
Stansfeld
,
Juha T.
Huiskonen
,
Paolo
Tammaro
,
Alessio
Accardi
,
Elisabeth P.
Carpenter
Diamond Proposal Number(s):
[10619, 15433]
Open Access
Abstract: Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.
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Sep 2019
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Krios I-Titan Krios I at Diamond
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Sarah V.
Faull
,
Andy M. C.
Lau
,
Chloe
Martens
,
Zainab
Ahdash
,
Kjetil
Hansen
,
Hugo
Yebenes
,
Carla
Schmidt
,
Fabienne
Beuron
,
Nora B.
Cronin
,
Edward
Morris
,
Argyris
Politis
Open Access
Abstract: Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.
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Sep 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14739, 16258]
Open Access
Abstract: Stl is a master repressor encoded by Staphylococcus aureus pathogenicity islands (SaPIs) that maintains integration of these elements in the bacterial chromosome. After infection or induction of a resident helper phage, SaPIs are de-repressed by specific interactions of phage proteins with Stl. SaPIs have evolved a fascinating mechanism to ensure their promiscuous transfer by targeting structurally unrelated proteins performing identically conserved functions for the phage. Here we decipher the molecular mechanism of this elegant strategy by determining the structure of SaPIbov1 Stl alone and in complex with two structurally unrelated dUTPases from different S. aureus phages. Remarkably, SaPIbov1 Stl has evolved different domains implicated in DNA and partner recognition specificity. This work presents the solved structure of a SaPI repressor protein and the discovery of a modular repressor that acquires multispecificity through domain recruiting. Our results establish the mechanism that allows widespread dissemination of SaPIs in nature.
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Aug 2019
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[15848]
Open Access
Abstract: Molecular crystals can be bent elastically by expansion or plastically by delamination into slabs that glide along slip planes. Here we report that upon bending, terephthalic acid crystals can undergo a mechanically induced phase transition without delamination and their overall crystal integrity is retained. Such plastically bent crystals act as bimorphs and their phase uniformity can be recovered thermally by taking the crystal over the phase transition temperature. This recovers the original straight shape and the crystal can be bent by a reverse thermal treatment, resulting in shape memory effects akin of those observed with some metal alloys and polymers. We anticipate that similar memory and restorative effects are common for other molecular crystals having metastable polymorphs. The results demonstrate the advantage of using intermolecular interactions to accomplish mechanically adaptive properties with organic solids that bridge the gap between mesophasic and inorganic materials in the materials property space.
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Aug 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Lucile
Moynie
,
Stefan
Milenkovic
,
Gaëtan L. A.
Mislin
,
Véronique
Gasser
,
Giuliano
Malloci
,
Etienne
Baco
,
Rory P.
Mccaughan
,
Malcolm G. P.
Page
,
Isabelle J.
Schalk
,
Matteo
Ceccarelli
,
James H.
Naismith
Open Access
Abstract: Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe3+ complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.
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Aug 2019
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744, 10627]
Open Access
Abstract: Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. Here, we determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, our structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. We demonstrate that semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.
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Aug 2019
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I05-ARPES
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H. F.
Yang
,
L. X.
Yang
,
Z. K.
Liu
,
Y.
Sun
,
C.
Chen
,
H.
Peng
,
M.
Schmidt
,
D.
Prabhakaran
,
B. A.
Bernevig
,
C.
Felser
,
B. H.
Yan
,
Y. L.
Chen
Diamond Proposal Number(s):
[19310, 18005]
Open Access
Abstract: Surface Fermi arcs (SFAs), the unique open Fermi-surfaces (FSs) discovered recently in topological Weyl semimetals (TWSs), are unlike closed FSs in conventional materials and can give rise to many exotic phenomena, such as anomalous SFA-mediated quantum oscillations, chiral magnetic effects, three-dimensional quantum Hall effect, non-local voltage generation and anomalous electromagnetic wave transmission. Here, by using in-situ surface decoration, we demonstrate successful manipulation of the shape, size and even the connections of SFAs in a model TWS, NbAs, and observe their evolution that leads to an unusual topological Lifshitz transition not caused by the change of the carrier concentration. The phase transition teleports the SFAs between different parts of the surface Brillouin zone. Despite the dramatic surface evolution, the existence of SFAs is robust and each SFA remains tied to a pair of Weyl points of opposite chirality, as dictated by the bulk topology.
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Aug 2019
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[18360]
Open Access
Abstract: Herpesviridae is a vast family of enveloped DNA viruses that includes eight distinct human pathogens, responsible for diseases that range from almost asymptomatic to severe and life-threatening. Epstein-Barr virus infects B-cells and epithelial cells, causing infectious mononucleosis, as well as a number of cancers. Epstein-Barr infection cannot be cured since neither vaccine nor antiviral drug treatments are available. All herpesviruses contain a linear double-stranded DNA genome, enclosed within an icosahedral capsid. Viral portal protein plays a key role in the procapsid assembly and DNA packaging. The portal is the entrance and exit pore for the viral genome, making it an attractive pharmacological target for the development of new antivirals. Here we present the atomic structure of the portal protein of Epstein-Barr virus, solved by cryo-electron microscopy at 3.5 Å resolution. The detailed architecture of this protein suggests that it plays a functional role in DNA retention during packaging.
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Aug 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Platelet recruitment to sites of blood vessel damage is highly dependent upon von Willebrand factor (VWF). VWF platelet-tethering function is proteolytically regulated by the metalloprotease ADAMTS13. Proteolysis depends upon shear-induced conformational changes in VWF that reveal the A2 domain cleavage site. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2 domain. Here we report through kinetic analyses that, in binding VWF, the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloprotease domain to facilitate proteolysis. The crystal structure of the ADAMTS13 metalloprotease to spacer domains reveals that the metalloprotease domain exhibits a latent conformation in which the active-site cleft is occluded supporting the requirement for an allosteric change to enable accommodation of the substrate. Our data demonstrate that VWF functions as both the activating cofactor and substrate for ADAMTS13.
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Aug 2019
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