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104 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6, 7, 8 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Novel and improved crystal structures of Haemophilus influenzae, E. coli and P. aeruginosa Penicillin-Binding-Protein 3 (PBP3) and N. gonorrhoeae PBP2: Towards a better understanding of β-lactam target-mediated resistance Dom Bellini , Lizbe Koekemoer , Hector Newman , Christopher G. Dowson Journal Of Molecular Biology DOI: 10.1016/j.jmb.2019.07.010 Show Abstract ▼ Abstract: Even with the emergence of antibiotic resistance, penicillin and the wider family of beta-lactams have remained the single most important family of antibiotics. The periasmic/extracytoplasmic targets of penicillin are a family of enzymes with a highly conserved catalytic activity involved in the final stage of bacterial cell wall (peptidoglycan, PG) biosynthesis. Named after their ability to bind penicillin, rather than their catalytic activity these key targets are called penicillin-binding proteins (PBPs).Resistance is predominantly mediated by reducing the target drug concentration via beta-lactamases, however, naturally transformable bacteria have also acquired target mediated resistance by inter-species recombination. Here we focus on structural based interpretations of amino acid alterations associated with the emergence of resistance within clinical isolates and include new PBP3 structures along with new, and improved, PBP-beta-lactam co-structures.	Jul 2019
B21-High Throughput SAXS	The molecular basis for apolipoprotein E4 as the major risk factor for late-onset Alzheimer's disease Ana-caroline Raulin , Lucas Kraft , Youssra K. Al-hilaly , Wei-feng Xue , John E. Mcgeehan , John R. Atack , Louise Serpell Journal Of Molecular Biology DOI: 10.1016/j.jmb.2019.04.019 Diamond Proposal Number(s): [14891] Show Abstract ▼ Abstract: Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerization state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small-angle x-ray scattering, analytical ultracentrifugation, circular dichroism, x-ray fiber diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerize to form wavy filaments, which do not share the characteristics of cross-β amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis, and these results open opportunities for uncovering new triggers for AD onset.	Apr 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	A cell-surface GH9 endo-glucanase coordinates with surface glycan binding proteins to mediate xyloglucan uptake in the gut symbiont Bacteroides ovatus Matthew H. Foley , Guillaume Déjean , Glyn R. Hemsworth , Gideon J. Davies , Harry Brumer , Nicole M. Koropatkin Journal Of Molecular Biology DOI: 10.1016/j.jmb.2019.01.008 Diamond Proposal Number(s): [7864] Show Abstract ▼ Abstract: Dietary fiber is an important food source for members of the human gut microbiome. Members of the dominant Bacteroidetes phylum capture diverse polysaccharides via the action of multiple cell surface proteins encoded within Polysaccharide Utilization Loci (PUL). The independent activities of PUL-encoded glycoside hydrolases (GH) and surface glycan-binding proteins (SGBPs) for the harvest of various glycans have been studied in detail, but how these proteins work together to coordinate uptake is poorly understood. Here, we combine genetic and biochemical approaches to discern the interplay between the BoGH9 endoglucanase and the xyloglucan-binding proteins SGBP-A and SGBP-B from the Bacteroides ovatus Xyloglucan Utilization Locus (XyGUL). The expression of BoGH9, a weakly active xyloglucanase in isolation, is required in a strain that expresses a non-binding version of SGBP-A (SGBP-A). The crystal structure of the BoGH9 enzyme suggests the molecular basis for its robust activity on mixed-linkage β-glucan compared to xyloglucan. Yet, catalytically inactive site-directed mutants of BoGH9 fail to complement the deletion of the active BoGH9 in a SGBP-A strain. We also find that SGBP-B is needed in an SGBP-A* background to support growth on xyloglucan, but that the non-binding SGBP-B* protein acts in a dominant negative manner to inhibit growth on xyloglucan. We postulate a model whereby the SGBP-A, SGBP-B and BoGH9 work together at the cell surface, likely within a discrete complex, and that xyloglucan binding by SGBP-B and BoGH9 may facilitate the orientation of the xyloglucan for transfer across the outer membrane.	Jan 2019
B21-High Throughput SAXS I03-Macromolecular Crystallography	A nucleotide-dependent conformational switch controls the polymerization of human IMP dehydrogenases to modulate their catalytic activity David Fernandez-justel , Rafael Nunez , Jaime Martin-benito , David Jimeno , Adrián González-lópez , Eva María Soriano , José Luis Revuelta , Ruben M. Buey Journal Of Molecular Biology DOI: 10.1016/j.jmb.2019.01.020 Diamond Proposal Number(s): [10121] Show Abstract ▼ Abstract: IMP dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo GTP biosynthetic pathway and plays essential roles in cell proliferation. As a clinical target, IMPDH has been studied for decades but it has only been within the last years that we are starting to understand the complexity of the mechanisms of its physiological regulation. Here, we report structural and functional insights into how adenine and guanine nucleotides control a conformational switch that modulates the assembly of the two human IMPDH enzymes into cytoophidia and allosterically regulates their catalytic activity. In vitro reconstituted micron-length cytoophidia-like structures show catalytic activity comparable to unassembled IMPDH but, in turn, are more resistant to GTP/GDP allosteric inhibition. Therefore, IMPDH cytoophidia formation facilitates the accumulation of high levels of guanine nucleotides when the cell requires it. Finally, we demonstrate that most of the IMPDH retinopathy-associated mutations abrogate GTP/GDP-induced allosteric inhibition and alter cytoophidia dynamics.	Jan 2019
B21-High Throughput SAXS	Regulation of human HSC70 ATPase and chaperone activities by Apg2: Role of the acidic subdomain Yovana Cabrera , Leire Dublang , José Angel Fernández-higuero , David Albesa-jove , Maria Lucas , Ana Rosa Viguera , Marcelo E. Guerin , Jose M. G. Vilar , Arturo Muga , Fernando Moro Journal Of Molecular Biology DOI: 10.1016/j.jmb.2018.11.026 Diamond Proposal Number(s): [15304] Show Abstract ▼ Abstract: Protein aggregate reactivation in metazoans is accomplished by the combined activity of Hsp70, Hsp40 and Hsp110 chaperones. Hsp110s support the refolding of aggregated polypeptides acting as specialized nucleotide exchange factors of Hsp70. We have studied how Apg2, one of the three human Hsp110s, regulates the activity of Hsc70 (HspA8), the constitutive Hsp70 in our cells. Apg2 shows a biphasic behavior: at low concentration, it stimulates the ATPase cycle of Hsc70, binding of the chaperone to protein aggregates and the refolding activity of the system, while it inhibits these three processes at high concentration. When the acidic subdomain of Apg2, a characteristic sequence present in the substrate binding domain of all Hsp110s, is deleted, the detrimental effects occur at lower concentration and are more pronounced, which concurs with an increase in the affinity of the Apg2 mutant for Hsc70. Our data support a mechanism in which Apg2 arrests the chaperone cycle through an interaction with Hsc70(ATP) that might lead to premature ATP dissociation before hydrolysis. In this line, the acidic subdomain might serve as a conformational switch to support dissociation of the Hsc70:Apg2 complex.	Dec 2018
B21-High Throughput SAXS	Structural variability of EspG chaperones from mycobacterial ESX-1, ESX-3 and ESX-5 Type VII secretion systems Anne T. Tuukkanen , Diana Freire , Sum Chan , Mark A. Arbing , Robert W. Reed , Timothy J. Evans , Grasilda Zenkeviciutė , Jennifer Kim , Sara Kahng , Michael R. Sawaya , Catherine T. Chaton , Matthias Wilmanns , David Eisenberg , Annabel H. A. Parret , Konstantin V. Korotkov Journal Of Molecular Biology DOI: 10.1016/j.jmb.2018.11.003 Diamond Proposal Number(s): [10231] Show Abstract ▼ Abstract: Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. Additionally, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3 and EspG5 chaperones using small-angle X-ray scattering (SAXS) reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3− PE5-PPE4 complex based on the SAXS analysis.	Nov 2018
I02-Macromolecular Crystallography	A new mechanism for high-affinity uptake of C4-dicarboxylates in bacteria revealed by the structure of rhodopseudomonas palustris matc (RPA3494), a periplasmic binding-protein of the tripartite tricarboxylate transporter (TTT) family Leonardo T. Rosa , Samuel R. Dix , John B. Rafferty , David J. Kelly Journal Of Molecular Biology DOI: 10.1016/j.jmb.2018.11.016 Diamond Proposal Number(s): [12788] Open Access Show Abstract ▼ Abstract: C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric-acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the Tripartite Tricarboxylate Transport (TTT) family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to L- and D-malate with Kd values of 27 and 21 nM respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (Kd 110 nM) and fumarate (Kd 400 nM). The 2.1 Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data, and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized TTT family, which are widely distributed in bacterial genomes but for which details of structure–function relationships and transport mechanisms have been lacking.	Nov 2018
Krios II-Titan Krios II at Diamond	Structure of mutant hepatitis B core protein capsids with premature secretion phenotype Bettina Böttcher , Michael Nassal Journal Of Molecular Biology DOI: 10.1016/j.jmb.2018.10.018 Diamond Proposal Number(s): [14580] Show Abstract ▼ Abstract: Hepatitis B virus is a major human pathogen that consists of a viral genome surrounded by an icosahedrally ordered core protein and a polymorphic, lipidic envelope that is densely packed with surface proteins. A point mutation in the core protein in which a phenylalanine at position 97 is exchanged for a smaller leucine leads to premature envelopment of the capsid before the genome maturation is fully completed. We have used electron cryo microscopy and image processing to investigate how the point mutation affects the structure of the capsid at 2.6–2.8 Å resolution. We found that in the mutant the smaller side chain at position 97 is displaced, increasing the size of an adjacent pocket in the centre of the spikes of the capsid. In the mutant this pocket is filled with an unknown density. Phosphorylation of serine residues in the unresolved C-terminal domain of the mutant leaves the structure of the ordered capsid largely unchanged. However, we were able to resolve several previously unresolved residues downstream of proline 144 that precede the phosphorylation-sites. These residues pack against the neighboring subunits and increase the inter-dimer contact suggesting that the C-termini play an important role in capsid stabilization and provide a much larger interaction interface than previously observed.	Oct 2018
I03-Macromolecular Crystallography	Cotranslational folding of a pentarepeat β-helix protein Luigi Notari , Markel Martinez-carranza , Jose Arcadio Farias-rico , Pal Stenmark , Gunnar Von Heijne Journal Of Molecular Biology DOI: 10.1016/j.jmb.2018.10.016 Diamond Proposal Number(s): [11265] Show Abstract ▼ Abstract: It is becoming increasingly clear that many proteins start to fold cotranslationally, before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, i.e., proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum – a homolog of the Fluoroquinolone Resistance Protein MfpA – using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling, and that folding starts when this unit is ~ 35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo, and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.	Oct 2018
I03-Macromolecular Crystallography	The FapF amyloid secretion transporter possesses an atypical asymmetric coiled coil Sarah L. Rouse , Fisentzos Stylianou , H. Y. Grace Wu , Jamie-lee Berry , Lee Sewell , R. Marc L. Morgan , Andrea C. Sauerwein , Steve Matthews Journal Of Molecular Biology DOI: 10.1016/j.jmb.2018.06.007 Diamond Proposal Number(s): [12579] Open Access Show Abstract ▼ Abstract: Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits, the fibres of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialised outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8 Å resolution. This domain forms a novel asymmetric trimeric coiled-coil that possesses a single buried tyrosine residue as well as a extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail.	Jun 2018

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