I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Alan
Cartmell
,
Elisabeth C.
Lowe
,
Arnaud
Basle
,
Susan J.
Firbank
,
Didier A.
Ndeh
,
Heath
Murray
,
Nicolas
Terrapon
,
Vincent
Lombard
,
Bernard
Henrissat
,
Jeremy E.
Turnbull
,
Mirjam
Czjzek
,
Harry J.
Gilbert
,
David N.
Bolam
Diamond Proposal Number(s):
[311, 9948]
Abstract: The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron, a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS, and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides, indicating that the model developed is of generic relevance to this important microbial community.
|
Jul 2017
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Amy J.
Glenwright
,
Karunakar R.
Pothula
,
Satya P.
Bhamidimarri
,
Dror S.
Chorev
,
Arnaud
Basle
,
Susan J.
Firbank
,
Hongjun
Zheng
,
Carol V.
Robinson
,
Mathias
Winterhalter
,
Ulrich
Kleinekathöfer
,
David N.
Bolam
,
Bert
Van Den Berg
Diamond Proposal Number(s):
[9948]
Abstract: The human large intestine is populated by a high density of microorganisms, collectively termed the colonic microbiota1, which has an important role in human health and nutrition2. The survival of microbiota members from the dominant Gram-negative phylum Bacteroidetes depends on their ability to degrade dietary glycans that cannot be metabolized by the host3. The genes encoding proteins involved in the degradation of specific glycans are organized into co-regulated polysaccharide utilization loci4, 5, 6, 7, 8, with the archetypal locus sus (for starch utilisation system) encoding seven proteins, SusA–SusG8, 9, 10. Glycan degradation mainly occurs intracellularly and depends on the import of oligosaccharides by an outer membrane protein complex composed of an extracellular SusD-like lipoprotein and an integral membrane SusC-like TonB-dependent transporter4, 5, 6, 7, 11, 12, 13. The presence of the partner SusD-like lipoprotein is the major feature that distinguishes SusC-like proteins from previously characterized TonB-dependent transporters. Many sequenced gut Bacteroides spp. encode over 100 SusCD pairs, of which the majority have unknown functions and substrate specificities3, 8, 14, 15. The mechanism by which extracellular substrate binding by SusD proteins is coupled to outer membrane passage through their cognate SusC transporter is unknown. Here we present X-ray crystal structures of two functionally distinct SusCD complexes purified from Bacteroides thetaiotaomicron and derive a general model for substrate translocation. The SusC transporters form homodimers, with each β-barrel protomer tightly capped by SusD. Ligands are bound at the SusC–SusD interface in a large solvent-excluded cavity. Molecular dynamics simulations and single-channel electrophysiology reveal a ‘pedal bin’ mechanism, in which SusD moves away from SusC in a hinge-like fashion in the absence of ligand to expose the substrate-binding site to the extracellular milieu. These data provide mechanistic insights into outer membrane nutrient import by members of the microbiota, an area of major importance for understanding human–microbiota symbiosis.
|
Jan 2017
|
|
I02-Macromolecular Crystallography
|
Open Access
Abstract: zinc and copper are required by proteins with very different functions, these metals can be delivered to cellular locations by homologous metal transporters within the same organism, as demonstrated by the cyanobacterial (Synechocystis PCC 6803) zinc exporter ZiaA and thylakoidal copper importer PacS. The N-terminal metal-binding domains of these transporters (ZiaAN and PacSN, respectively) have related ferredoxin folds also found in the metallochaperone Atx1, which delivers copper to PacS, but differ in the residues found in their M/IXCXXC metal-binding motifs.
|
Sep 2013
|
|
I04-Macromolecular Crystallography
|
Open Access
Abstract: The copper metallochaperone Atx1 and the N-terminal metalbinding domain of a copper-transporting ATP-ase can form tight Zn(II)-mediated hetero-complexes in both cyanobacteria and humans. Copper and zinc homeostasis could be linked by metal binding to these CXXC-containing proteins.
|
Sep 2013
|
|
I04-Macromolecular Crystallography
|
Abstract: Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing bacteria (methanotrophs) that have been linked to the substantial copper needs of these environmentally important microorganisms. The only characterized mbs are those from Methylosinus trichosporium OB3b and Methylocystis strain SB2. M. trichosporium OB3b produces a second mb (mb-Met), which is missing the C-terminal Met residue from the full-length form (FL-mb). The as-isolated copper-loaded mbs bind Cu(I). The absence of the Met has little influence on the structure of the Cu(I) site, and both molecules mediate switchover from the soluble iron methane mono-oxygenase to the particulate copper-containing enzyme in M. trichosporium OB3b cells. Cu(II) is reduced in the presence of the mbs under our experimental conditions, and the disulfide plays no role in this process. The Cu(I) affinities of these molecules are extremely high with values of (6?7) × 1020 M?1 determined at pH ? 8.0. The affinity for Cu(I) is 1 order of magnitude lower at pH 6.0. The reduction potentials of copper-loaded FL-mb and mb-Met are 640 and 590 mV respectively, highlighting the strong preference for Cu(I) and indicating different Cu(II) affinities for the two forms. Cleavage of the disulfide bridge results in a decrease in the Cu(I) affinity to ?9 × 1018 M?1 at pH 7.5. The two thiolates can also bind Cu(I), albeit with much lower affinity (? 3 × 1015 M?1 at pH 7.5). The high affinity of mbs for Cu(I) is consistent with a physiological role in copper uptake and protection.
|
Jan 2011
|
|
I03-Macromolecular Crystallography
|
Abstract: Acopper-trafficking pathway was found to enable Cu2 occupancy
of a soluble periplasm protein, CucA, even when competing
Zn2 is abundant in the periplasm. Here, we solved the
structure of CucA (a new cupin) and found that binding of Cu2,
but not Zn2, quenches the fluorescence of Trp165, which is
adjacent to the metal site. Using this fluorescence probe, we
established that CucA becomes partly occupied by Zn2 following
exposure to equimolar Zn2 and Cu2. Cu2-CucA is more
thermodynamically stable than Zn2-CucA but k(Zn3Cu)exchange
is slow, raising questions about how the periplasm contains
solely the Cu2 form. We discovered that a copper-trafficking
pathway involving two copper transporters (CtaA and PacS)
and a metallochaperone (Atx1) is obligatory for Cu2-CucA to
accumulate in the periplasm. There was negligible CucA protein
in the periplasm of ctaA cells, but the abundance of cucA transcripts
was unaltered. Crucially, ctaA cells overaccumulate
low Mr copper complexes in the periplasm, and purified
apoCucA can readily acquire Cu2 from ctaA periplasm
extracts, but in vivo apoCucA fails to come into contact with
these periplasmic copper pools. Instead, copper traffics via a
cytoplasmic pathway that is coupled to CucA translocation to
the periplasm.
|
Oct 2010
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
|
Abstract: Molecular systems have evolved to permit the safe delivery of copper. Despite extensive studies, many copper site structures involved in copper homeostasis, even for the well-studied metallochaperone Atx1, remain unresolved. Cyanobacteria import copper to their thylakoid compartments for use in photosynthesis and respiration and possess an Atx1 that we show can adopt multiple oligomeric states when metalated, capable of binding up to four copper ions. Two-copper- and four-copper-loaded dimers exist in solution at low micromolar concentrations, and head-to-head and side-to-side arrangements, respectively, can be crystallized, with the latter binding a [Cu4{?2-S?(Cys)}4Cl2]2? cluster. The His61Tyr mutation on loop 5 weakens head-to-head dimerization, yet a side-to-side dimer binding a similar cluster as in the wild-type protein, but with phenolate coordination, is present. The cognate metal-binding domains (MBDs) of the P-type ATPases CtaA and PacS, which are proposed to donate copper to and accept copper from Atx1, respectively, are monomeric in the presence of copper. The structure of the MBD of Cu(I)-PacS shows a crystallographic trimer arrangement around a [Cu3{?2-S?(Cys)}3{S?(Cys)}3]2? cluster that is very similar to that found for an alternate form of the His61Tyr Atx1 mutant. Copper transfer from the MBD of CtaA to Atx1 is favorable, but delivery from Atx1 to the MBD of PacS is strongly dependent upon the dimeric form of Atx1. A copper-induced switch in Atx1 dimer structure may have a regulatory role with cluster formation helping to buffer copper.
|
Aug 2010
|
|
I04-Macromolecular Crystallography
|
Abstract: Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describe uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein−DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase−base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at position 2 explains the stimulation of the polymerase 3′−5′ proofreading exonuclease, observed with deaminated bases at this location. A β-hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double-stranded DNA. This denatures the two complementary primer bases and directs the resulting 3′ single-stranded extension toward the exonuclease active site. Finally, the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using nonpolar isosteres. Affinity for both 2,4-difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine, respectively, is strongly diminished, suggesting polar protein−base contacts are important. However, residual interaction with 2,4-difluorobenzene is seen, confirming a role for shape recognition.
|
Jun 2010
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Abstract: The intestinal microbiota impacts many facets of human health and is associated with human diseases. Diet impacts microbiota composition, yet mechanisms that link dietary changes to microbiota alterations remain ill-defined. Here we elucidate the basis of Bacteroides proliferation in response to fructans, a class of fructose-based dietary polysaccharides. Structural and genetic analysis disclosed a fructose-binding, hybrid two-component signaling sensor that controls the fructan utilization locus in Bacteroides thetaiotaomicron. Gene content of this locus differs among Bacteroides species and dictates the specificity and breadth of utilizable fructans. BT1760, an extracellular β2-6 endo-fructanase, distinguishes B. thetaiotaomicron genetically and functionally, and enables the use of the β2-6-linked fructan levan. The genetic and functional differences between Bacteroides species are predictive of in vivo competitiveness in the presence of dietary fructans. Gene sequences that distinguish species' metabolic capacity serve as potential biomarkers in microbiomic datasets to enable rational manipulation of the microbiota via diet.
|
Jun 2010
|
|
I03-Macromolecular Crystallography
|
Cedric
Montanier
,
Alicia
Lammerts Van Bueren
,
Claire
Dumon
,
James E.
Flint
,
Márcia
Correia
,
Jose A.
Prates
,
Susan
Firbank
,
Rick
Lewis
,
Gilles G.
Grondin
,
Mariana G.
Ghinet
,
Tracey M.
Gloster
,
Cecile
Herve
,
J. Paul
Knox
,
Brian G.
Talbot
,
Johan
Turkenburg
,
Janne
Kerovuo
,
Ryszard
Brzezinski
,
Carlos M. G. A.
Fontes
,
Gideon J.
Davies
,
Alisdair B.
Boraston
,
Harry J.
Gilbert
Abstract: Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-?-d-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or ?4,5-anhydrogalaturonic acid (?4,5-GalA). ?4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.
|
Mar 2009
|
|
