I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Anna
Barwinska-sendra
,
Yuritzi M.
Garcia
,
Kacper M.
Sendra
,
Arnaud
Basle
,
Eilidh S.
Mackenzie
,
Emma
Tarrant
,
Patrick
Card
,
Leandro C.
Tabares
,
Cédric
Bicep
,
Sun
Un
,
Thomas E.
Kehl-fie
,
Kevin J.
Waldron
Diamond Proposal Number(s):
[7864, 9948, 18598]
Open Access
Abstract: Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal’s redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.
|
Jun 2020
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Mariusz
Madej
,
Joshua B. R.
White
,
Zuzanna
Nowakowska
,
Shaun
Rawson
,
Carsten
Scavenius
,
Jan J.
Enghild
,
Grzegorz P.
Bereta
,
Karunakar
Pothula
,
Ulrich
Kleinekathoefer
,
Arnaud
Basle
,
Neil A.
Ranson
,
Jan
Potempa
,
Bert
Van Den Berg
Diamond Proposal Number(s):
[13587]
Abstract: Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how these peptides enter the cell is not clear. Here, we identify RagAB as the outer-membrane importer for these peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex, with the RagB substrate-binding surface-anchored lipoprotein forming a closed lid on the RagA TonB-dependent transporter. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a ‘pedal bin’ mechanism of nutrient uptake. Together with mutagenesis, peptide-binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic, selective outer-membrane oligopeptide-acquisition machine that is essential for the efficient utilization of proteinaceous nutrients by P. gingivalis.
|
May 2020
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Many biologists are now routinely seeking to determine the three-dimensional structures of their proteins of choice, illustrating the importance of this knowledge, but also of the simplification and streamlining of structure-determination processes. Despite the fact that most software packages offer simple pipelines, for the non-expert navigating the outputs and understanding the key aspects can be daunting. Here, the structure determination of the type IV pili (TFP) protein PilA1 from Clostridioides difficile is used to illustrate the different steps involved, the key decision criteria and important considerations when using the most common pipelines and software. Molecular-replacement pipelines within CCP4i2 are presented to illustrate the more commonly used processes. Previous knowledge of the biology and structure of TFP pilins, particularly the presence of a long, N-terminal α-helix required for pilus formation, allowed informed decisions to be made during the structure-determination strategy. The PilA1 structure was finally successfully determined using ARCIMBOLDO and the ab initio MR strategy used is described.
|
Mar 2020
|
|
|
|
Open Access
Abstract: This study describes a method to estimate the likelihood of success in determining a macromolecular structure by X-ray crystallography and experimental single-wavelength anomalous dispersion (SAD) or multiple-wavelength anomalous dispersion (MAD) phasing based on initial data-processing statistics and sample crystal properties. Such a predictive tool can rapidly assess the usefulness of data and guide the collection of an optimal data set. The increase in data rates from modern macromolecular crystallography beamlines, together with a demand from users for real-time feedback, has led to pressure on computational resources and a need for smarter data handling. Statistical and machine-learning methods have been applied to construct a classifier that displays 95% accuracy for training and testing data sets compiled from 440 solved structures. Applying this classifier to new data achieved 79% accuracy. These scores already provide clear guidance as to the effective use of computing resources and offer a starting point for a personalized data-collection assistant.
|
Mar 2020
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the ‘sulfation problem’ is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.
|
Jan 2020
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Justina
Briliūtė
,
Paulina A.
Urbanowicz
,
Ana S.
Luis
,
Arnaud
Basle
,
Neil
Paterson
,
Osmond
Rebello
,
Jenifer
Hendel
,
Didier A.
Ndeh
,
Elisabeth C.
Lowe
,
Eric C.
Martens
,
Daniel I. R.
Spencer
,
David N.
Bolam
,
Lucy I.
Crouch
Diamond Proposal Number(s):
[13587, 18598]
Abstract: Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.
|
Jun 2019
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[13587]
Abstract: The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiome. The selection pressures in this environment have spurred the evolution of a complex reservoir of microbial genes encoding carbohydrate-active enzymes (CAZymes). Previously, we have shown that the human gut bacterium Bacteroides thetaiotaomicron (Bt) can depolymerize the most structurally complex glycan, the plant pectin rhamnogalacturonan II (RGII), commonly found in the human diet. Previous investigation of the RGIIdegrading apparatus in Bt identified BT0997 as a new CAZyme family, classified as glycoside hydrolase 138 (GH138). The mechanism of substrate recognition by GH138, however, remains unclear. Here, using synthetic substrates and biochemical assays, we show that BT0997 targets the D-galacturonic acid-α-1,2-L-rhamnose linkage in chain A of RGII and that it absolutely requires the presence of a second D-galacturonic acid side chain (linked β-1,3 to L-rhamnose) for activity. NMR analysis revealed that BT0997 operates through a double-displacement, retaining mechanism. We also report the crystal structure of a BT0997 homolog, BPA0997 from Bacteroides paurosaccharolyticus, in complex with ligands at 1.6 Å resolution. The structure disclosed that the enzyme comprises four domains, including a catalytic TIM (α/β)8 barrel. Characterization of several BT0997 variants identified Glu-294 and Glu361 as the catalytic acid/base and nucleophile, respectively, and we observed a chloride ion close to the active site. The three-dimensional structure and bioinformatic analysis revealed that two arginines, Arg-332 and Arg-521, are key specificity determinants of BT0997 in targeting D-galacturonic acid residues. In summary, our study reports the first structural and mechanistic analyses of GH138 enzymes.
|
Mar 2019
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Alan
Cartmell
,
Jose
Muñoz-muñoz
,
Jonathon A.
Briggs
,
Didier A.
Ndeh
,
Elisabeth C.
Lowe
,
Arnaud
Basle
,
Nicolas
Terrapon
,
Katherine
Stott
,
Tiaan
Heunis
,
Joe
Gray
,
Li
Yu
,
Paul
Dupree
,
Pearl Z.
Fernandes
,
Sayali
Shah
,
Spencer J.
Williams
,
Aurore
Labourel
,
Matthias
Trost
,
Bernard
Henrissat
,
Harry J.
Gilbert
Diamond Proposal Number(s):
[1960, 7854, 9948]
Abstract: Glycans are major nutrients for the human gut microbiota (HGM). Arabinogalactan proteins (AGPs) comprise a heterogenous group of plant glycans in which a β1,3-galactan backbone and β1,6-galactan side chains are conserved. Diversity is provided by the variable nature of the sugars that decorate the galactans. The mechanisms by which nutritionally relevant AGPs are degraded in the HGM are poorly understood. Here we explore how the HGM organism Bacteroides thetaiotaomicron metabolizes AGPs. We propose a sequential degradative model in which exo-acting glycoside hydrolase (GH) family 43 β1,3-galactanases release the side chains. These oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or GH. We identified two GHs that establish two previously undiscovered GH families. The crystal structures of the exo-β1,3-galactanases identified a key specificity determinant and departure from the canonical catalytic apparatus of GH43 enzymes. Growth studies of Bacteroidetes spp. on complex AGP revealed 3 keystone organisms that facilitated utilization of the glycan by 17 recipient bacteria, which included B. thetaiotaomicron. A surface endo-β1,3-galactanase, when engineered into B. thetaiotaomicron, enabled the bacterium to utilize complex AGPs and act as a keystone organism.
|
Nov 2018
|
|
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[9948]
Abstract: Acinetobacter baumannii is becoming a major threat to human health due to its multidrug resistance. This is owing in a large part to the low permeability of its outer membrane (OM), which prevents high internal antibiotic concentrations and makes antibiotic-resistance mechanisms more effective. To exploit OM channels as potential delivery vehicles for future antibiotics, structural information is required. One abundant OM protein in A. baumannii is Omp33. This protein has been reported to be important for the in vivo fitness and virulence of A. baumannii, but its structure is not known. Here, the X-ray crystal structure of Omp33 is reported at a resolution of 2.1 Å. Omp33 has a 14-β-stranded barrel without stable extracellular loop constrictions. Instead, an extended and unusual periplasmic turn connecting β-strands 2 and 3 is present, which folds into the pore lumen and completely blocks the aqueous channel. The Omp33 structure helps in understanding how A. baumannii OM proteins contribute to the low permeability of the cell envelope of this bacterium and suggests that Omp33 might function as a gated channel.
|
Sep 2018
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[9948, 13587]
Abstract: The outer membranes (OM) of many Gram-negative bacteria contain general porins, which form nonspecific, large-diameter channels for the diffusional uptake of small molecules required for cell growth and function. While the porins of Enterobacteriaceae (e.g., E. coli OmpF and OmpC) have been extensively characterized structurally and biochemically, much less is known about their counterparts in Vibrionaceae. Vibrio cholerae, the causative agent of cholera, has two major porins, OmpU and OmpT, for which no structural information is available despite their importance for the bacterium. Here we report high-resolution X-ray crystal structures of V. cholerae OmpU and OmpT complemented with molecular dynamics simulations. While similar overall to other general porins, the channels of OmpU and OmpT have unusual constrictions that create narrower barriers for small-molecule permeation and change the internal electric fields of the channels. Together with electrophysiological and in vitro transport data, our results illuminate small-molecule uptake within the Vibrionaceae.
|
Apr 2018
|
|
