Publications by authors named "Keith S Wilson"

108 Publications

Epitope Mapping of Exposed Tegument and Alimentary Tract Proteins Identifies Putative Antigenic Targets of the Attenuated Schistosome Vaccine.

Front Immunol 2020 3;11:624613. Epub 2021 Mar 3.

York Biomedical Research Institute, University of York, York, United Kingdom.

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against . Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.
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http://dx.doi.org/10.3389/fimmu.2020.624613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7982949PMC
March 2021

Fungal GH25 muramidases: New family members with applications in animal nutrition and a crystal structure at 0.78Å resolution.

PLoS One 2021 12;16(3):e0248190. Epub 2021 Mar 12.

Department of Chemistry, Structural Biology Laboratory, The University of York, York, United Kingdom.

Muramidases/lysozymes hydrolyse the peptidoglycan component of the bacterial cell wall. They are found in many of the glycoside hydrolase (GH) families. Family GH25 contains muramidases/lysozymes, known as CH type lysozymes, as they were initially discovered in the Chalaropsis species of fungus. The characterized enzymes from GH25 exhibit both β-1,4-N-acetyl- and β-1,4-N,6-O-diacetylmuramidase activities, cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties in the carbohydrate backbone of bacterial peptidoglycan. Here, a set of fungal GH25 muramidases were identified from a sequence search, cloned and expressed and screened for their ability to digest bacterial peptidoglycan, to be used in a commercial application in chicken feed. The screen identified the enzyme from Acremonium alcalophilum JCM 736 as a suitable candidate for this purpose and its relevant biochemical and biophysical and properties are described. We report the crystal structure of the A. alcalophilum enzyme at atomic, 0.78 Å resolution, together with that of its homologue from Trichobolus zukalii at 1.4 Å, and compare these with the structures of homologues. GH25 enzymes offer a new solution in animal feed applications such as for processing bacterial debris in the animal gut.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248190PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954357PMC
March 2021

Predicting protein model correctness in Coot using machine learning.

Acta Crystallogr D Struct Biol 2020 Aug 27;76(Pt 8):713-723. Epub 2020 Jul 27.

Department of Chemistry, University of York, York YO10 5DD, United Kingdom.

Manually identifying and correcting errors in protein models can be a slow process, but improvements in validation tools and automated model-building software can contribute to reducing this burden. This article presents a new correctness score that is produced by combining multiple sources of information using a neural network. The residues in 639 automatically built models were marked as correct or incorrect by comparing them with the coordinates deposited in the PDB. A number of features were also calculated for each residue using Coot, including map-to-model correlation, density values, B factors, clashes, Ramachandran scores, rotamer scores and resolution. Two neural networks were created using these features as inputs: one to predict the correctness of main-chain atoms and the other for side chains. The 639 structures were split into 511 that were used to train the neural networks and 128 that were used to test performance. The predicted correctness scores could correctly categorize 92.3% of the main-chain atoms and 87.6% of the side chains. A Coot ML Correctness script was written to display the scores in a graphical user interface as well as for the automatic pruning of chains, residues and side chains with low scores. The automatic pruning function was added to the CCP4i2 Buccaneer automated model-building pipeline, leading to significant improvements, especially for high-resolution structures.
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http://dx.doi.org/10.1107/S2059798320009080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397494PMC
August 2020

Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis.

PeerJ 2020 26;8:e9408. Epub 2020 Jun 26.

Structural Biology and NMR Laboratory, Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen N, Denmark.

Background: Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown.

Methods: Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals.

Results: The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.
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http://dx.doi.org/10.7717/peerj.9408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323712PMC
June 2020

The C-Type Lysozyme from the upper Gastrointestinal Tract of the Stinkbird.

Int J Mol Sci 2019 Nov 6;20(22). Epub 2019 Nov 6.

Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, UK.

Muramidases/lysozymes are important bio-molecules, which cleave the glycan backbone in the peptidoglycan polymer found in bacterial cell walls. The glycoside hydrolase (GH) family 22 C-type lysozyme, from the folivorous bird (stinkbird), was expressed in , and a set of variants was produced. All variants were enzymatically active, including those designed to probe key differences between the Hoatzin enzyme and Hen Egg White lysozyme. Four variants showed improved thermostability at pH 4.7, compared to the wild type. The X-ray structure of the enzyme was determined in the apo form and in complex with chitin oligomers. Bioinformatic analysis of avian GH22 amino acid sequences showed that they separate out into three distinct subgroups (chicken-like birds, sea birds and other birds). The Hoatzin is found in the "other birds" group and we propose that this represents a new cluster of avian upper-gut enzymes.
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http://dx.doi.org/10.3390/ijms20225531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887759PMC
November 2019

Structural and Functional Characterization of Three Novel Fungal Amylases with Enhanced Stability and pH Tolerance.

Int J Mol Sci 2019 Oct 3;20(19). Epub 2019 Oct 3.

York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, UK.

Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from and . The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the enzyme was identified, which might be part of a starch interaction site. In addition, amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.
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http://dx.doi.org/10.3390/ijms20194902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801514PMC
October 2019

Novel Inhibitory Function of the Lipase Propeptide and Three-Dimensional Structures of Its Complexes with the Enzyme.

ACS Omega 2019 Jun 7;4(6):9964-9975. Epub 2019 Jun 7.

York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, U.K.

Many proteins are synthesized as precursors, with propeptides playing a variety of roles such as assisting in folding or preventing them from being active within the cell. While the precise role of the propeptide in fungal lipases is not completely understood, it was previously reported that mutations in the propeptide region of the lipase have an influence on the activity of the mature enzyme, stressing the importance of the amino acid composition of this region. We here report two structures of this enzyme in complex with its propeptide, which suggests that the latter plays a role in the correct maturation of the enzyme. Most importantly, we demonstrate that the propeptide shows inhibition of lipase activity in standard lipase assays and propose that an important role of the propeptide is to ensure that the enzyme is not active during its expression pathway in the original host.
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http://dx.doi.org/10.1021/acsomega.9b00612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648591PMC
June 2019

Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Secretomes Using Activity-Based Probes.

ACS Cent Sci 2019 Jun 24;5(6):1067-1078. Epub 2019 May 24.

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands.

Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and -β-1,4-xylanases in the secretomes of , by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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http://dx.doi.org/10.1021/acscentsci.9b00221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598175PMC
June 2019

Crystal structure and substrate interactions of an unusual fungal non-CBM carrying GH26 endo-β-mannanase from Yunnania penicillata.

Sci Rep 2019 02 19;9(1):2266. Epub 2019 Feb 19.

Novozymes A/S, Krogshøjvej 36, 2880, Bagsværd, Denmark.

Endo-β(1 → 4)-mannanases (endomannanases) catalyse degradation of β-mannans, an abundant class of plant polysaccharides. This study investigates structural features and substrate binding of YpenMan26A, a non-CBM carrying endomannanase from Yunnania penicillata. Structural and sequence comparisons to other fungal family GH26 endomannanases showed high sequence similarities and conserved binding residues, indicating that fungal GH26 endomannanases accommodate galactopyranosyl units in the -3 and -2 subsites. Two striking amino acid differences in the active site were found when the YpenMan26A structure was compared to a homology model of Wsp.Man26A from Westerdykella sp. and the sequences of nine other fungal GH26 endomannanases. Two YpenMan26A mutants, W110H and D37T, inspired by differences observed in Wsp.Man26A, produced a shift in how mannopentaose bound across the active site cleft and a decreased affinity for galactose in the -2 subsite, respectively, compared to YpenMan26A. YpenMan26A was moreover found to have a flexible surface loop in the position where PansMan26A from Podospora anserina has an α-helix (α9) which interacts with its family 35 CBM. Sequence alignment inferred that the core structure of fungal GH26 endomannanases differ depending on the natural presence of this type of CBM. These new findings have implications for selecting and optimising these enzymes for galactomannandegradation.
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http://dx.doi.org/10.1038/s41598-019-38602-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381184PMC
February 2019

The structure of the AliC GH13 α-amylase from Alicyclobacillus sp. reveals the accommodation of starch branching points in the α-amylase family.

Acta Crystallogr D Struct Biol 2019 Jan 4;75(Pt 1):1-7. Epub 2019 Jan 4.

York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.

α-Amylases are glycoside hydrolases that break the α-1,4 bonds in starch and related glycans. The degradation of starch is rendered difficult by the presence of varying degrees of α-1,6 branch points and their possible accommodation within the active centre of α-amylase enzymes. Given the myriad industrial uses for starch and thus also for α-amylase-catalysed starch degradation and modification, there is considerable interest in how different α-amylases might accommodate these branches, thus impacting on the potential processing of highly branched post-hydrolysis remnants (known as limit dextrins) and societal applications. Here, it was sought to probe the branch-point accommodation of the Alicyclobacillus sp. CAZy family GH13 α-amylase AliC, prompted by the observation of a molecule of glucose in a position that may represent a branch point in an acarbose complex solved at 2.1 Å resolution. Limit digest analysis by two-dimensional NMR using both pullulan (a regular linear polysaccharide of α-1,4, α-1,4, α-1,6 repeating trisaccharides) and amylopectin starch showed how the Alicyclobacillus sp. enzyme could accept α-1,6 branches in at least the -2, +1 and +2 subsites, consistent with the three-dimensional structures with glucosyl moieties in the +1 and +2 subsites and the solvent-exposure of the -2 subsite 6-hydroxyl group. Together, the work provides a rare insight into branch-point acceptance in these industrial catalysts.
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http://dx.doi.org/10.1107/S2059798318014900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333287PMC
January 2019

Mimicking salmochelin S1 and the interactions of its Fe(III) complex with periplasmic iron siderophore binding proteins CeuE and VctP.

J Inorg Biochem 2019 01 19;190:75-84. Epub 2018 Oct 19.

Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, UK. Electronic address:

A mimic of the tetradentate stealth siderophore salmochelin S1, was synthesised, characterised and shown to form Fe(III) complexes with ligand-to-metal ratios of 1:1 and 3:2. Circular dichroism spectroscopy confirmed that the periplasmic binding proteins CeuE and VctP of Campylobacter jejuni and Vibrio cholerae, respectively, bind the Fe(III) complex of the salmochelin mimic by preferentially selecting Λ-configured Fe(III) complexes. Intrinsic fluorescence quenching studies revealed that VctP binds Fe(III) complexes of the mimic and structurally-related catecholate ligands, such as enterobactin, bis(2, 3-dihydroxybenzoyl-l-serine) and bis(2, 3-dihydroxybenzoyl)-1, 5-pentanediamine with higher affinity than does CeuE. Both CeuE and VctP display a clear preference for the tetradentate bis(catecholates) over the tris(catecholate) siderophore enterobactin. These findings are consistent with reports that V. cholerae and C. jejuni utilise the enterobactin hydrolysis product bis(2, 3-dihydroxybenzoyl)-O-seryl serine for the acquisition of Fe(III) and suggest that the role of salmochelin S1 in the iron uptake of enteric pathogens merits further investigation.
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http://dx.doi.org/10.1016/j.jinorgbio.2018.10.008DOI Listing
January 2019

Structure of a Talaromyces pinophilus GH62 arabinofuranosidase in complex with AraDNJ at 1.25 Å resolution.

Acta Crystallogr F Struct Biol Commun 2018 Aug 26;74(Pt 8):490-495. Epub 2018 Jul 26.

York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.

The enzymatic hydrolysis of complex plant biomass is a major societal goal of the 21st century in order to deliver renewable energy from nonpetroleum and nonfood sources. One of the major problems in many industrial processes, including the production of second-generation biofuels from lignocellulose, is the presence of `hemicelluloses' such as xylans which block access to the cellulosic biomass. Xylans, with a polymeric β-1,4-xylose backbone, are frequently decorated with acetyl, glucuronyl and arabinofuranosyl `side-chain' substituents, all of which need to be removed for complete degradation of the xylan. As such, there is interest in side-chain-cleaving enzymes and their action on polymeric substrates. Here, the 1.25 Å resolution structure of the Talaromyces pinophilus arabinofuranosidase in complex with the inhibitor AraDNJ, which binds with a K of 24 ± 0.4 µM, is reported. Positively charged iminosugars are generally considered to be potent inhibitors of retaining glycosidases by virtue of their ability to interact with both acid/base and nucleophilic carboxylates. Here, AraDNJ shows good inhibition of an inverting enzyme, allowing further insight into the structural basis for arabinoxylan recognition and degradation.
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http://dx.doi.org/10.1107/S2053230X18000250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096477PMC
August 2018

Structural insight into industrially relevant glucoamylases: flexible positions of starch-binding domains.

Acta Crystallogr D Struct Biol 2018 05 26;74(Pt 5):463-470. Epub 2018 Apr 26.

Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, England.

Glucoamylases are one of the most important classes of enzymes in the industrial degradation of starch biomass. They consist of a catalytic domain and a carbohydrate-binding domain (CBM), with the latter being important for the interaction with the polymeric substrate. Whereas the catalytic mechanisms and structures of the individual domains are well known, the spatial arrangement of the domains with respect to each other and its influence on activity are not fully understood. Here, the structures of three industrially used fungal glucoamylases, two of which are full length, have been crystallized and determined. It is shown for the first time that the relative orientation between the CBM and the catalytic domain is flexible, as they can adopt different orientations independently of ligand binding, suggesting a role as an anchor to increase the contact time and the relative concentration of substrate near the active site. The flexibility in the orientations of the two domains presented a considerable challenge for the crystallization of the enzymes.
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http://dx.doi.org/10.1107/S2059798318004989DOI Listing
May 2018

From crystal to structure with CCP4.

Acta Crystallogr D Struct Biol 2018 Feb 1;74(Pt 2):67. Epub 2018 Feb 1.

York Structural Biology Laboratory, University of York, Heslington, York, YO10 5DD, UK.

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http://dx.doi.org/10.1107/S2059798317017557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947770PMC
February 2018

Recent developments in MrBUMP: better search-model preparation, graphical interaction with search models, and solution improvement and assessment.

Acta Crystallogr D Struct Biol 2018 03 6;74(Pt 3):167-182. Epub 2018 Mar 6.

Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, England.

Increasing sophistication in molecular-replacement (MR) software and the rapid expansion of the PDB in recent years have allowed the technique to become the dominant method for determining the phases of a target structure in macromolecular X-ray crystallography. In addition, improvements in bioinformatic techniques for finding suitable homologous structures for use as MR search models, combined with developments in refinement and model-building techniques, have pushed the applicability of MR to lower sequence identities and made weak MR solutions more amenable to refinement and improvement. MrBUMP is a CCP4 pipeline which automates all stages of the MR procedure. Its scope covers everything from the sourcing and preparation of suitable search models right through to rebuilding of the positioned search model. Recent improvements to the pipeline include the adoption of more sensitive bioinformatic tools for sourcing search models, enhanced model-preparation techniques including better ensembling of homologues, and the use of phase improvement and model building on the resulting solution. The pipeline has also been deployed as an online service through CCP4 online, which allows its users to exploit large bioinformatic databases and coarse-grained parallelism to speed up the determination of a possible solution. Finally, the molecular-graphics application CCP4mg has been combined with MrBUMP to provide an interactive visual aid to the user during the process of selecting and manipulating search models for use in MR. Here, these developments in MrBUMP are described with a case study to explore how some of the enhancements to the pipeline and to CCP4mg can help to solve a difficult case.
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http://dx.doi.org/10.1107/S2059798318003455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947758PMC
March 2018

Structural Characterization of Acidic M17 Leucine Aminopeptidases from the TriTryps and Evaluation of Their Role in Nutrient Starvation in .

mSphere 2017 Jul-Aug;2(4). Epub 2017 Aug 16.

Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Armilla, Granada, Spain.

Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in , where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from , , and ) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in , strongly suggesting that the enzyme is not required for the growth of this parasite either or . In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in , LAP-A may participate in protein degradation associated with nutrient depletion. Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both and , an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction.
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http://dx.doi.org/10.1128/mSphere.00226-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557676PMC
August 2017

Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM siderophore analogues of varied linker length.

Sci Rep 2017 04 6;7:45941. Epub 2017 Apr 6.

Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, UK.

Bacteria use siderophores to mediate the transport of essential Fe(III) into the cell. In Campylobacter jejuni the periplasmic binding protein CeuE, an integral part of the Fe(III) transport system, has adapted to bind tetradentate siderophores using a His and a Tyr side chain to complete the Fe(III) coordination. A series of tetradentate siderophore mimics was synthesized in which the length of the linker between the two iron-binding catecholamide units was increased from four carbon atoms (4-LICAM) to five, six and eight (5-, 6-, 8-LICAM, respectively). Co-crystal structures with CeuE showed that the inter-planar angles between the iron-binding catecholamide units in the 5-, 6- and 8-LICAM structures are very similar (111°, 110° and 110°) and allow for an optimum fit into the binding pocket of CeuE, the inter-planar angle in the structure of 4-LICAM is significantly smaller (97°) due to restrictions imposed by the shorter linker. Accordingly, the protein-binding affinity was found to be slightly higher for 5- compared to 4-LICAM but decreases for 6- and 8-LICAM. The optimum linker length of five matches that present in natural siderophores such as enterobactin and azotochelin. Site-directed mutagenesis was used to investigate the relative importance of the Fe(III)-coordinating residues H227 and Y288.
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http://dx.doi.org/10.1038/srep45941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382913PMC
April 2017

The structure of a calcium-dependent phosphoinositide-specific phospholipase C from Pseudomonas sp. 62186, the first from a Gram-negative bacterium.

Acta Crystallogr D Struct Biol 2017 Jan 1;73(Pt 1):32-44. Epub 2017 Jan 1.

York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, England.

Bacterial phosphoinositide-specific phospholipases C (PI-PLCs) are the smallest members of the PI-PLC family, which includes much larger mammalian enzymes responsible for signal transduction as well as enzymes from protozoan parasites, yeast and plants. Eukaryotic PI-PLCs have calcium in the active site, but this is absent in the known structures of Gram-positive bacteria, where its role is instead played by arginine. In addition to their use in a number of industrial applications, the bacterial enzymes attract special interest because they can serve as convenient models of the catalytic domains of eukaryotic enzymes for in vitro activity studies. Here, the structure of a PI-PLC from Pseudomonas sp. 62186 is reported, the first from a Gram-negative bacterium and the first of a native bacterial PI-PLC with calcium present in the active site. Solution of the structure posed particular problems owing to the low sequence identity of available homologous structures. Its dependence on calcium for catalysis makes this enzyme a better model for studies of the mammalian PI-PLCs than the previously used calcium-independent bacterial PI-PLCs.
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http://dx.doi.org/10.1107/S2059798316019616DOI Listing
January 2017

Carbohydrate structure: the rocky road to automation.

Curr Opin Struct Biol 2017 06 9;44:39-47. Epub 2016 Dec 9.

York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK. Electronic address:

With the introduction of intuitive graphical software, structural biologists who are not experts in crystallography are now able to build complete protein or nucleic acid models rapidly. In contrast, carbohydrates are in a wholly different situation: scant automation exists, with manual building attempts being sometimes toppled by incorrect dictionaries or refinement problems. Sugars are the most stereochemically complex family of biomolecules and, as pyranose rings, have clear conformational preferences. Despite this, all refinement programs may produce high-energy conformations at medium to low resolution, without any support from the electron density. This problem renders the affected structures unusable in glyco-chemical terms. Bringing structural glycobiology up to 'protein standards' will require a total overhaul of the methodology. Time is of the essence, as the community is steadily increasing the production rate of glycoproteins, and electron cryo-microscopy has just started to image them in precisely that resolution range where crystallographic methods falter most.
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http://dx.doi.org/10.1016/j.sbi.2016.11.011DOI Listing
June 2017

Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function.

Biochim Biophys Acta Proteins Proteom 2017 Jan 28;1865(1):20-27. Epub 2016 Sep 28.

Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark. Electronic address:

Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions.
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http://dx.doi.org/10.1016/j.bbapap.2016.09.016DOI Listing
January 2017

Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei.

Mol Microbiol 2016 11 31;102(3):365-385. Epub 2016 Aug 31.

Instituto de Parasitología y Biomedicina "López-Neyra", Consejo Superior de Investigaciones Científicas, Granada, Spain.

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.
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http://dx.doi.org/10.1111/mmi.13467DOI Listing
November 2016

Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product.

Proc Natl Acad Sci U S A 2016 May 9;113(21):5850-5. Epub 2016 May 9.

Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom;

To acquire essential Fe(III), bacteria produce and secrete siderophores with high affinity and selectivity for Fe(III) to mediate its uptake into the cell. Here, we show that the periplasmic binding protein CeuE of Campylobacter jejuni, which was previously thought to bind the Fe(III) complex of the hexadentate siderophore enterobactin (Kd ∼ 0.4 ± 0.1 µM), preferentially binds the Fe(III) complex of the tetradentate enterobactin hydrolysis product bis(2,3-dihydroxybenzoyl-l-Ser) (H5-bisDHBS) (Kd = 10.1 ± 3.8 nM). The protein selects Λ-configured [Fe(bisDHBS)](2-) from a pool of diastereomeric Fe(III)-bisDHBS species that includes complexes with metal-to-ligand ratios of 1:1 and 2:3. Cocrystal structures show that, in addition to electrostatic interactions and hydrogen bonding, [Fe(bisDHBS)](2-) binds through coordination of His227 and Tyr288 to the iron center. Similar binding is observed for the Fe(III) complex of the bidentate hydrolysis product 2,3-dihydroxybenzoyl-l-Ser, [Fe(monoDHBS)2](3-) The mutation of His227 and Tyr288 to noncoordinating residues (H227L/Y288F) resulted in a substantial loss of affinity for [Fe(bisDHBS)](2-) (Kd ∼ 0.5 ± 0.2 µM). These results suggest a previously unidentified role for CeuE within the Fe(III) uptake system of C. jejuni, provide a molecular-level understanding of the underlying binding pocket adaptations, and rationalize reports on the use of enterobactin hydrolysis products by C. jejuni, Vibrio cholerae, and other bacteria with homologous periplasmic binding proteins.
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http://dx.doi.org/10.1073/pnas.1520829113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889360PMC
May 2016

Three-dimensional structures of two heavily N-glycosylated Aspergillus sp. family GH3 β-D-glucosidases.

Acta Crystallogr D Struct Biol 2016 Feb 28;72(Pt 2):254-65. Epub 2016 Jan 28.

York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.

The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo- and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant β-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. β-D-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 β-D-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley β-D-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the D-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy (4)C1 chair conformation and should be of general application to refinements of other crystal structures containing O- or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal β-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.
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http://dx.doi.org/10.1107/S2059798315024237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756609PMC
February 2016

Privateer: software for the conformational validation of carbohydrate structures.

Nat Struct Mol Biol 2015 Nov;22(11):833-4

Department of Chemistry, University of York, York, UK.

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http://dx.doi.org/10.1038/nsmb.3115DOI Listing
November 2015

The substrate-binding protein in bacterial ABC transporters: dissecting roles in the evolution of substrate specificity.

Biochem Soc Trans 2015 Oct;43(5):1011-7

Department of Biology, University of York, Wentworth Way, York Y010 5YW, U.K.

ATP-binding cassette (ABC) transporters, although being ubiquitous in biology, often feature a subunit that is limited primarily to bacteria and archaea. This subunit, the substrate-binding protein (SBP), is a key determinant of the substrate specificity and high affinity of ABC uptake systems in these organisms. Most prokaryotes have many SBP-dependent ABC transporters that recognize a broad range of ligands from metal ions to amino acids, sugars and peptides. Herein, we review the structure and function of a number of more unusual SBPs, including an ABC transporter involved in the transport of rare furanose forms of sugars and an SBP that has evolved to specifically recognize the bacterial cell wall-derived murein tripeptide (Mtp). Both these examples illustrate that subtle changes in binding-site architecture, including changes in side chains not directly involved in ligand co-ordination, can result in significant alteration of substrate range in novel and unpredictable ways.
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http://dx.doi.org/10.1042/BST20150135DOI Listing
October 2015

Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major.

PLoS Negl Trop Dis 2015 May 15;9(5):e0003781. Epub 2015 May 15.

Structural Biology Laboratory, Department of Chemistry, University of York, York, United Kingdom.

Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s(-1) and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5'-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs.
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http://dx.doi.org/10.1371/journal.pntd.0003781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433323PMC
May 2015

The three-dimensional structure of the cellobiohydrolase Cel7A from Aspergillus fumigatus at 1.5 Å resolution.

Acta Crystallogr F Struct Biol Commun 2015 Jan 1;71(Pt 1):114-20. Epub 2015 Jan 1.

Department of Chemistry, University of York, York Structural Biology Laboratory, York YO10 5DD, England.

The enzymatic degradation of plant cell-wall cellulose is central to many industrial processes, including second-generation biofuel production. Key players in this deconstruction are the fungal cellobiohydrolases (CBHs), notably those from family GH7 of the carbohydrate-active enzymes (CAZY) database, which are generally known as CBHI enzymes. Here, three-dimensional structures are reported of the Aspergillus fumigatus CBHI Cel7A solved in uncomplexed and disaccharide-bound forms at resolutions of 1.8 and 1.5 Å, respectively. The product complex with a disaccharide in the +1 and +2 subsites adds to the growing three-dimensional insight into this family of industrially relevant biocatalysts.
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http://dx.doi.org/10.1107/S2053230X14027307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304761PMC
January 2015

Three-dimensional structure of a variant `Termamyl-like' Geobacillus stearothermophilus α-amylase at 1.9 Å resolution.

Acta Crystallogr F Struct Biol Commun 2015 Jan 1;71(Pt 1):66-70. Epub 2015 Jan 1.

York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.

The enzyme-catalysed degradation of starch is central to many industrial processes, including sugar manufacture and first-generation biofuels. Classical biotechnological platforms involve steam explosion of starch followed by the action of endo-acting glycoside hydrolases termed α-amylases and then exo-acting α-glucosidases (glucoamylases) to yield glucose, which is subsequently processed. A key enzymatic player in this pipeline is the `Termamyl' class of bacterial α-amylases and designed/evolved variants thereof. Here, the three-dimensional structure of one such Termamyl α-amylase variant based upon the parent Geobacillus stearothermophilus α-amylase is presented. The structure has been solved at 1.9 Å resolution, revealing the classical three-domain fold stabilized by Ca2+ and a Ca2+-Na+-Ca2+ triad. As expected, the structure is similar to the G. stearothermophilus α-amylase but with main-chain deviations of up to 3 Å in some regions, reflecting both the mutations and differing crystal-packing environments.
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http://dx.doi.org/10.1107/S2053230X14026508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304751PMC
January 2015

Thermodynamic and structural investigation of the specific SDS binding of Humicola insolens cutinase.

Protein Sci 2014 Aug 16;23(8):1023-35. Epub 2014 Jun 16.

Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000, Aalborg, Denmark.

The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (Ka ≈ 10(5) M(-1) ). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.
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http://dx.doi.org/10.1002/pro.2489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4116652PMC
August 2014

Analysis of an industrial production suspension of Bacillus lentus subtilisin crystals by powder diffraction: a powerful quality-control tool.

Acta Crystallogr D Biol Crystallogr 2014 Apr 21;70(Pt 4):1115-23. Epub 2014 Mar 21.

Department of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800 Kgs. Lyngby, Denmark.

A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Å and a = 52.72, b = 57.13, c = 65.86 Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Å with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.
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http://dx.doi.org/10.1107/S1399004714001497DOI Listing
April 2014