I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Aspergillus fumigatus is a human opportunistic fungal pathogen whose cell wall protects it from the extracellular environment, including host defense responses. Chitin, an essential component of the fungal cell wall, is synthesized from UDP-GlcNAc produced in the hexosamine biosynthetic pathway. Because this pathway is critical for fungal cell wall integrity, the hexosamine biosynthesis enzymes represent potential targets of antifungal drugs. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. GNA1 deletion resulted in loss of fungal viability and disruption of the cell wall, phenotypes that could be rescued by exogenous GlcNAc, the product of the Gna1 enzyme. In a murine model of aspergillosis, the Δgna1 mutant strain exhibited attenuated virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the Gna1 active site and can be optimized to a selective sub-micromolar binder. Taken together, we have provided genetic, structural, and chemical evidence that Gna1 is an antifungal target in A. fumigatus.
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Apr 2020
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B21-High Throughput SAXS
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Catherine R.
Back
,
Victoria A.
Higman
,
Kristian
Levay
,
Viren V.
Patel
,
Alice E.
Parnell
,
Daniel
Frankel
,
Howard F.
Jenkinson
,
Steven G.
Burston
,
Matthew P.
Crump
,
Angela H.
Nobbs
,
Paul R.
Race
Diamond Proposal Number(s):
[13400, 12342]
Abstract: The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to seventeen ~100-amino-acid-long repeat domains (RDs). Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1-17, domains 1–17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all β-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1-17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.
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Mar 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15806]
Open Access
Abstract: MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo, as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP-bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site sub-pocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1 catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway.
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Mar 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Abstract: Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family (Bs164) from the human gut symbiont Bacteroides salyersiae. Previous reports of this enzyme indicated that it has α-mannosidase activity, however we conclusively show that it cleaves only β-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of wild-type and mutant Bs164, and multi-angle light scattering we found that it is a trimeric retaining β-mannosidase, that is susceptible to several known mannosidase inhibitors. X-ray crystallography revealed the structure of Bs164 – the first known structure of a GH164 – at 1.91 Å resolution. Bs164 is composed of three domains: a (β/α)8 barrel, a trimerization domain and a β-sandwich domain, representing a previously unobserved structural fold for β-mannosidases. Structures of Bs164 at 1.80-2.55 Å resolution in complex with the inhibitors noeuromycin, mannoimidazole or DNP 2-deoxy-2-fluoro-mannose reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu297) and acid/base residue (Glu160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.
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Dec 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433]
Abstract: Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors, and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the “nude” phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC), that is different from the more widely-recognized forkhead (FKH) sequence RYAAAYA (R = purine, Y = pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5’-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.
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Dec 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Abstract: The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain’s dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the wild-type globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain’s dimerization. Biophysical experiments revealed that whereas the isolated wild-type globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.
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Dec 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15304]
Abstract: Bacillus subtilis PdaC (BsPdaC) is a membrane-bound, multidomain peptidoglycan N-deacetylase acting on N-acetylmuramic (Mur-NAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. BsPdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, BsPdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the BsPdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that BsPdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, BsPdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase SpPgdaA than with other MurNAc deacetylases. Therefore, we used ligand-docking simulations to analyze the dual GlcNAc- and MurNAc-binding specificities of BsPdaC and compared them with those of SpPgdA and BsPdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. BsPdaC retains the conserved Asp–His–His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. BsPdaC contains short loops similar to those in SpPgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases.
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Nov 2019
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B21-High Throughput SAXS
Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[16619, 17773]
Open Access
Abstract: Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multiangle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies.
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Nov 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Shiqi
Ji
,
Samuel R.
Dix
,
Adli A.
Aziz
,
Svetlana E.
Sedelnikova
,
Patrick J.
Baker
,
John B.
Rafferty
,
Per A.
Bullough
,
Svetomir B.
Tzokov
,
Jon
Agirre
,
Fu-li
Li
,
David W.
Rice
Diamond Proposal Number(s):
[17773]
Abstract: Alginate is a polymer containing two uronic acid epimers, β-d-mannuronate (M) and α-l-guluronate (G), and is a major component of brown seaweed that is depolymerized by alginate lyases. These enzymes have diverse specificity, cleaving the chain with endo- or exotype activity and with differential selectivity for the sequence of M or G at the cleavage site. Dp0100 is a 201-kDa multi-modular, broad-specificity endotype alginate lyase from the marine thermophile Defluviitalea phaphyphila, which uses brown algae as a carbon source, converting it to ethanol, and bioinformatics analysis suggested that its catalytic domain represents a new polysaccharide lyase family, PLxx. The structure of the Dp0100 catalytic domain, determined at 2.07 Å resolution, revealed that it comprises three regions strongly resembling those of the exotype lyase families PL15 and PL17. The conservation of key catalytic histidine and tyrosine residues belonging to the latter suggest these enzymes share mechanistic similarities. A complex of Dp0100 with a pentasaccharide, M5, showed that the oligosaccharide is located in subsites –2, –1, +1, +2, and +3 in a long, deep canyon open at both ends, explaining the endotype activity of this lyase. This contrasted with the hindered binding sites of the exotype enzymes, which are blocked such that only one sugar moiety can be accommodated at the –1 position in the catalytic site. The biochemical and structural analyses of Dp0100, the first for this new class of endotype alginate lyases, has furthered our understanding of the structure-function and evolutionary relationships within this important class of enzymes.
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Oct 2019
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I02-Macromolecular Crystallography
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Bruce J.
Maclachlan
,
Garry
Dolton
,
Athanasios
Papakyriakou
,
Alexander
Greenshields-watson
,
Georgina H.
Mason
,
Andrea
Schauenburg
,
Matthieu
Besneux
,
Barbara
Szomolay
,
Tim
Elliott
,
Andrew K.
Sewell
,
Awen
Gallimore
,
Pierre
Rizkallah
,
David K.
Cole
,
Andrew
Godkin
Diamond Proposal Number(s):
[10462]
Abstract: CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide flanking residues (PFRs) can extend beyond the limits of the HLA-binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence TCR affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak T-cell receptor (TCR) binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak-affinity TCR-pHLA-II interactions.
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Oct 2019
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