Publications by authors named "Edward Lowe"

76 Publications

Reconstitution and Structural Analysis of a HECT Ligase-Ubiquitin Complex via an Activity-Based Probe.

ACS Chem Biol 2021 09 17;16(9):1615-1621. Epub 2021 Aug 17.

Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

Ubiquitin activity-based probes have proven invaluable in elucidating structural mechanisms in the ubiquitin system by stabilizing transient macromolecular complexes of deubiquitinases, ubiquitin-activating enzymes, and the assemblies of ubiquitin-conjugating enzymes with ubiquitin ligases of the RING-Between-RING and RING-Cysteine-Relay families. Here, we demonstrate that an activity-based probe, ubiquitin-propargylamine, allows for the preparative reconstitution and structural analysis of the interactions between ubiquitin and certain HECT ligases. We present a crystal structure of the ubiquitin-linked HECT domain of HUWE1 that defines a catalytically critical conformation of the C-terminal tail of the ligase for the transfer of ubiquitin to an acceptor protein. Moreover, we observe that ubiquitin-propargylamine displays selectivity among HECT domains, thus corroborating the notion that activity-based probes may provide entry points for the development of specific, active site-directed inhibitors and reporters of HECT ligase activities.
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http://dx.doi.org/10.1021/acschembio.1c00433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8453484PMC
September 2021

Toxin import through the antibiotic efflux channel TolC.

Nat Commun 2021 07 30;12(1):4625. Epub 2021 Jul 30.

Department of Biochemistry, University of Oxford, Oxford, UK.

Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae, binds TolC of a related species (K. quasipneumoniae) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins.
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http://dx.doi.org/10.1038/s41467-021-24930-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324772PMC
July 2021

Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein.

Structure 2021 Sep 28;29(9):1014-1028.e8. Epub 2021 Apr 28.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK. Electronic address:

The kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Taken together, these results provide structural insights into the unconventional kinetoplastid kinetochore protein KKT4.
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http://dx.doi.org/10.1016/j.str.2021.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8443799PMC
September 2021

Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport.

mBio 2020 03 10;11(2). Epub 2020 Mar 10.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria. Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.
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http://dx.doi.org/10.1128/mBio.03230-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064778PMC
March 2020

Crystal structure of the catalytic C-lobe of the HECT-type ubiquitin ligase E6AP.

Protein Sci 2020 06 5;29(6):1550-1554. Epub 2020 Feb 5.

Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.

The HECT-type ubiquitin ligase E6AP (UBE3A) is critically involved in several neurodevelopmental disorders and human papilloma virus-induced cervical tumorigenesis; the structural mechanisms underlying the activity of this crucial ligase, however, are incompletely understood. Here, we report a crystal structure of the C-terminal lobe ("C-lobe") of the catalytic domain of E6AP that reveals two molecules in a domain-swapped, dimeric arrangement. Interestingly, the molecular hinge that enables this structural reorganization with respect to the monomeric fold coincides with the active-site region. While such dimerization is unlikely to occur in the context of full-length E6AP, we noticed a similar domain swap in a crystal structure of the isolated C-lobe of another HECT-type ubiquitin ligase, HERC6. This may point to conformational strain in the active-site region of HECT-type ligases with possible implications for catalysis. SIGNIFICANCE STATEMENT: The HECT-type ubiquitin ligase E6AP has key roles in human papilloma virus-induced cervical tumorigenesis and certain neurodevelopmental disorders. Here, we present a crystal structure of the C-terminal, catalytic lobe of E6AP, providing basic insight into the conformational properties of this functionally critical region of HECT-type ligases.
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http://dx.doi.org/10.1002/pro.3832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255509PMC
June 2020

Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons.

Cell 2020 01 9;180(2):323-339.e19. Epub 2020 Jan 9.

Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK. Electronic address:

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.
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http://dx.doi.org/10.1016/j.cell.2019.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978801PMC
January 2020

High-Throughput PIXE as an Essential Quantitative Assay for Accurate Metalloprotein Structural Analysis: Development and Application.

J Am Chem Soc 2020 01 20;142(1):185-197. Epub 2019 Dec 20.

Department of Biochemistry , University of Oxford , South Parks Road , Oxford OX1 3QU , United Kingdom.

Metalloproteins comprise over one-third of proteins, with approximately half of all enzymes requiring metal to function. Accurate identification of these metal atoms and their environment is a prerequisite to understanding biological mechanism. Using ion beam analysis through particle induced X-ray emission (PIXE), we have quantitatively identified the metal atoms in 30 previously structurally characterized proteins using minimal sample volume and a high-throughput approach. Over half of these metals had been misidentified in the deposited structural models. Some of the PIXE detected metals not seen in the models were explainable as artifacts from promiscuous crystallization reagents. For others, using the correct metal improved the structural models. For multinuclear sites, anomalous diffraction signals enabled the positioning of the correct metals to reveal previously obscured biological information. PIXE is insensitive to the chemical environment, but coupled with experimental diffraction data deposited alongside the structural model it enables validation and potential remediation of metalloprotein models, improving structural and, more importantly, mechanistic knowledge.
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http://dx.doi.org/10.1021/jacs.9b09186DOI Listing
January 2020

"Whatever I Have to Do That's Right:" Culture and the Precariousness of Personhood in a Poor Urban Neighborhood.

Authors:
Edward D Lowe

Ethos 2018 Sep 4;46(3):311-329. Epub 2018 Sep 4.

Associate Professor of Anthropology at Soka University of America.

This article presents a person-centered case study of one woman's struggles to realize a meaningful sense of personhood in a low-income urban neighborhood in Milwaukee, Wisconsin. An analysis of longitudinal ethnographic data for this case reveals how everyday aspirations toward a morally resonant lived-sense of personhood were informed by a core assemblage of three cultural models: "providing" and "being there" as a parent and doing so within a framework of "defensive individualism". This assemblage of cultural models was particularly compelling because of a combination of the embodied residue of childhood experiences and moments of "moral breakdown" in adult life. The experiences of moral breakdown were particularly meaningful because recurrent episodes of material hardship that constantly threatened to upend past efforts to realize a meaningful sense of personhood in everyday life and, in turn, generated a constant effort to reclaim and repair the symbolic markers of an achieved personhood that had been lost. These observations point to a precariousness of personhood that seemed to further motivate an investment in a self-definition in terms of this combination of cultural models.
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http://dx.doi.org/10.1111/etho.12205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223309PMC
September 2018

Pathological macromolecular crystallographic data affected by twinning, partial-disorder and exhibiting multiple lattices for testing of data processing and refinement tools.

Sci Rep 2018 10 5;8(1):14876. Epub 2018 Oct 5.

Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

Twinning is a crystal growth anomaly, which has posed a challenge in macromolecular crystallography (MX) since the earliest days. Many approaches have been used to treat twinned data in order to extract structural information. However, in most cases it is usually simpler to rescreen for new crystallization conditions that yield an untwinned crystal form or, if possible, collect data from non-twinned parts of the crystal. Here, we report 11 structures of engineered variants of the E. coli enzyme N-acetyl-neuraminic lyase which, despite twinning and incommensurate modulation, have been successfully indexed, solved and deposited. These structures span a resolution range of 1.45-2.30 Å, which is unusually high for datasets presenting such lattice disorders in MX and therefore these data provide an excellent test set for improving and challenging MX data processing programs.
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http://dx.doi.org/10.1038/s41598-018-32962-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173773PMC
October 2018

Structures of Teneurin adhesion receptors reveal an ancient fold for cell-cell interaction.

Nat Commun 2018 03 14;9(1):1079. Epub 2018 Mar 14.

Department of Biochemistry, Oxford University, OX1 3QU, Oxford, UK.

Teneurins are ancient cell-cell adhesion receptors that are vital for brain development and synapse organisation. They originated in early metazoan evolution through a horizontal gene transfer event when a bacterial YD-repeat toxin fused to a eukaryotic receptor. We present X-ray crystallography and cryo-EM structures of two Teneurins, revealing a ~200 kDa extracellular super-fold in which eight sub-domains form an intricate structure centred on a spiralling YD-repeat shell. An alternatively spliced loop, which is implicated in homophilic Teneurin interaction and specificity, is exposed and thus poised for interaction. The N-terminal side of the shell is 'plugged' via a fibronectin-plug domain combination, which defines a new class of YD proteins. Unexpectedly, we find that these proteins are widespread amongst modern bacteria, suggesting early metazoan receptor evolution from a distinct class of proteins, which today includes both bacterial proteins and eukaryotic Teneurins.
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http://dx.doi.org/10.1038/s41467-018-03460-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851990PMC
March 2018

The N-Terminal Region of Fibrillin-1 Mediates a Bipartite Interaction with LTBP1.

Structure 2017 08 29;25(8):1208-1221.e5. Epub 2017 Jun 29.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

Fibrillin-1 (FBN1) mutations associated with Marfan syndrome lead to an increase in transforming growth factor β (TGF-β) activation in connective tissues resulting in pathogenic changes including aortic dilatation and dissection. Since FBN1 binds latent TGF-β binding proteins (LTBPs), the major reservoir of TGF-β in the extracellular matrix (ECM), we investigated the structural basis for the FBN1/LTBP1 interaction. We present the structure of a four-domain FBN1 fragment, EGF2-EGF3-Hyb1-cbEGF1 (FBN1), which reveals a near-linear domain organization. Binding studies demonstrate a bipartite interaction between a C-terminal LTBP1 fragment and FBN1, which lies adjacent to the latency-associated propeptide (LAP)/TGF-β binding site of LTBP1. Modeling of the binding interface suggests that, rather than interacting along the longitudinal axis, LTBP1 anchors itself to FBN1 using two independent epitopes. As part of this mechanism, a flexible pivot adjacent to the FBN1/LTBP1 binding site allows LTBP1 to make contacts with different ECM networks while presumably facilitating a force-induced/traction-based TGF-β activation mechanism.
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http://dx.doi.org/10.1016/j.str.2017.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548924PMC
August 2017

Investigation of the mycobacterial enzyme HsaD as a potential novel target for anti-tubercular agents using a fragment-based drug design approach.

Br J Pharmacol 2017 Jul 9;174(14):2209-2224. Epub 2017 May 9.

Faculty of Science, Engineering and Computing, Kingston University London, Kingston upon Thames, UK.

Background And Purpose: With the emergence of extensively drug-resistant tuberculosis, there is a need for new anti-tubercular drugs that work through novel mechanisms of action. The meta cleavage product hydrolase, HsaD, has been demonstrated to be critical for the survival of Mycobacterium tuberculosis in macrophages and is encoded in an operon involved in cholesterol catabolism, which is identical in M. tuberculosis and M. bovis BCG.

Experimental Approach: We generated a mutant strain of M. bovis BCG with a deletion of hsaD and tested its growth on cholesterol. Using a fragment based approach, over 1000 compounds were screened by a combination of differential scanning fluorimetry, NMR spectroscopy and enzymatic assay with pure recombinant HsaD to identify potential inhibitors. We used enzymological and structural studies to investigate derivatives of the inhibitors identified and to test their effects on growth of M. bovis BCG and M. tuberculosis.

Key Results: The hsaD deleted strain was unable to grow on cholesterol as sole carbon source but did grow on glucose. Of seven chemically distinct 'hits' from the library, two chemical classes of fragments were found to bind in the vicinity of the active site of HsaD by X-ray crystallography. The compounds also inhibited growth of M. tuberculosis on cholesterol. The most potent inhibitor of HsaD was also found to be the best inhibitor of mycobacterial growth on cholesterol-supplemented minimal medium.

Conclusions And Implications: We propose that HsaD is a novel therapeutic target, which should be fully exploited in order to design and discover new anti-tubercular drugs.

Linked Articles: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.
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http://dx.doi.org/10.1111/bph.13810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481647PMC
July 2017

High-Throughput Screening Assay Datasets from the PubChem Database.

Chem Inform 2017 26;3(1). Epub 2017 Apr 26.

Department of Chemistry, Pharmacology and Biomedical Informatics, Center for Structural Biology, Institute of Chemical Biology, Vanderbilt University, Nashville, USA.

Availability of high-throughput screening (HTS) data in the public domain offers great potential to foster development of ligand-based computer-aided drug discovery (LB-CADD) methods crucial for drug discovery efforts in academia and industry. LB-CADD method development depends on high-quality HTS assay data, i.e., datasets that contain both active and inactive compounds. These active compounds are hits from primary screens that have been tested in concentration-response experiments and where the target-specificity of the hits has been validated through suitable secondary screening experiments. Publicly available HTS repositories such as PubChem often provide such data in a convoluted way: compounds that are classified as inactive need to be extracted from the primary screening record. However, compounds classified as active in the primary screening record are not suitable as a set of active compounds for LB-CADD experiments due to high false-positive rate. A suitable set of actives can be derived by carefully analysing results in often up to five or more assays that are used to confirm and classify the activity of compounds. These assays, in part, build on each other. However, often not all hit compounds from the previous screen have been tested. Sometimes a compound can be classified as 'active', though its meaning is 'inactive' on the target of interest as it is 'active' on a different target protein. Here, a curation process of hierarchically related confirmatory screens is illustrated based on two specifically chosen protein use-cases. The subsequent re-upload procedure into PubChem is described for the findings of those two scenarios. Further, we provide nine publicly accessible high quality datasets for future LB-CADD method development that provide a common baseline for comparison of future methods to the scientific community. We also provide a protocol researchers can follow to upload additional datasets for benchmarking.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962024PMC
April 2017

Development of tools to automate quantitative analysis of radiation damage in SAXS experiments.

J Synchrotron Radiat 2017 01 1;24(Pt 1):63-72. Epub 2017 Jan 1.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

Biological small-angle X-ray scattering (SAXS) is an increasingly popular technique used to obtain nanoscale structural information on macromolecules in solution. However, radiation damage to the samples limits the amount of useful data that can be collected from a single sample. In contrast to the extensive analytical resources available for macromolecular crystallography (MX), there are relatively few tools to quantitate radiation damage for SAXS, some of which require a significant level of manual characterization, with the potential of leading to conflicting results from different studies. Here, computational tools have been developed to automate and standardize radiation damage analysis for SAXS data. RADDOSE-3D, a dose calculation software utility originally written for MX experiments, has been extended to account for the cylindrical geometry of the capillary tube, the liquid composition of the sample and the attenuation of the beam by the capillary material to allow doses to be calculated for many SAXS experiments. Furthermore, a library has been written to visualize and explore the pairwise similarity of frames. The calculated dose for the frame at which three subsequent frames are determined to be dissimilar is defined as the radiation damage onset threshold (RDOT). Analysis of RDOTs has been used to compare the efficacy of radioprotectant compounds to extend the useful lifetime of SAXS samples. Comparison of the RDOTs shows that, for radioprotectant compounds at 5 and 10 mM concentration, glycerol is the most effective compound. However, at 1 and 2 mM concentrations, dithiothreitol (DTT) appears to be most effective. Our newly developed visualization library contains methods that highlight the unusual radiation damage results given by SAXS data collected using higher concentrations of DTT: these observations should pave the way to the development of more sophisticated frame merging strategies.
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http://dx.doi.org/10.1107/S1600577516015083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5182020PMC
January 2017

Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals.

Proc Natl Acad Sci U S A 2016 08 26;113(32):E4630-8. Epub 2016 Jul 26.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;

The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme's quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme's activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.
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http://dx.doi.org/10.1073/pnas.1604463113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987793PMC
August 2016

Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.

J Mol Biol 2015 Aug 26;427(17):2852-66. Epub 2015 Jul 26.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups.
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http://dx.doi.org/10.1016/j.jmb.2015.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4548480PMC
August 2015

Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs.

J Enzyme Inhib Med Chem 2016 Aug 29;31(4):551-62. Epub 2015 May 29.

b Department of Chemistry , University of South Florida , Tampa , FL , USA , and.

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals.
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http://dx.doi.org/10.3109/14756366.2015.1046064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801743PMC
August 2016

BCL::SAXS: GPU accelerated Debye method for computation of small angle X-ray scattering profiles.

Proteins 2015 Aug 1;83(8):1500-12. Epub 2015 Jul 1.

Department of Biomedical Informatics, Vanderbilt University, Nashville, Tennessee, 37235.

Small angle X-ray scattering (SAXS) is an experimental technique used for structural characterization of macromolecules in solution. Here, we introduce BCL::SAXS--an algorithm designed to replicate SAXS profiles from rigid protein models at different levels of detail. We first show our derivation of BCL::SAXS and compare our results with the experimental scattering profile of hen egg white lysozyme. Using this protein we show how to generate SAXS profiles representing: (1) complete models, (2) models with approximated side chain coordinates, and (3) models with approximated side chain and loop region coordinates. We evaluated the ability of SAXS profiles to identify a correct protein topology from a non-redundant benchmark set of proteins. We find that complete SAXS profiles can be used to identify the correct protein by receiver operating characteristic (ROC) analysis with an area under the curve (AUC) > 99%. We show how our approximation of loop coordinates between secondary structure elements improves protein recognition by SAχS for protein models without loop regions and side chains. Agreement with SAXS data is a necessary but not sufficient condition for structure determination. We conclude that experimental SAXS data can be used as a filter to exclude protein models with large structural differences from the native.
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http://dx.doi.org/10.1002/prot.24838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797635PMC
August 2015

Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one.

Arch Biochem Biophys 2015 Jul 14;577-578:24-34. Epub 2015 May 14.

Division of Mathematical and Physical Sciences, Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA. Electronic address:

Tyrosinase (EC 1.14.18.1) catalyzes the monophenolase and diphenolase reaction associated with vertebrate pigmentation and fruit/vegetable browning. Tyrosinase is an oxygen-dependent, dicopper enzyme that has three states: Emet, Eoxy, and Edeoxy. The diphenolase activity can be carried out by both the met and the oxy states of the enzyme while neither mono- nor diphenolase activity results from the deoxy state. In this study, the oxidative cyclocondensation of 2-aminophenol (OAP) to the corresponding 2-aminophenoxazin-3-one (APX) by mushroom tyrosinase was investigated. Using a combination of various steady- and pre-steady state methodologies, we have investigated the kinetic and chemical mechanism of this reaction. The kcat for OAP is 75 ± 2s(-1), K(OAP)M = 1.8 ± 0.2mM, K(O2)M =25 ± 4 μM with substrates binding in a steady-state preferred fashion. Stopped flow and global analysis support a model where OAP preferentially binds to the oxy form over the met (k7 ≫ k1). For the met form, His269 and His61 are the proposed bases, while the oxy form uses the copper-peroxide and His61 for the sequential deprotonation of anilinic and phenolic hydrogens. Solvent KIEs show proton transfer to be increasingly rate limiting for kcat/K(OAP)M as [O2] → 0 μM (1.38 ± 0.06) decreasing to 0.83 ± 0.03 as [O2] → ∞ reflecting a partially rate limiting μ-OH bond cleavage (E met) and formation (E oxy) following protonation in the transition state. The coupling and cyclization reactions of o-quinone imine and OAP pass through a phenyliminocyclohexadione intermediate to APX, forming at a rate of 6.91 ± 0.03 μM(-1)s(-1) and 2.59E-2 ± 5.31E-4s(-1). Differences in reactivity attributed to the anilinic moiety of OAP with o-diphenols are discussed.
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http://dx.doi.org/10.1016/j.abb.2015.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456232PMC
July 2015

Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor.

Elife 2014 Dec 12;3:e05553. Epub 2014 Dec 12.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.
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http://dx.doi.org/10.7554/eLife.05553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383175PMC
December 2014

Structural conservation despite huge sequence diversity allows EPCR binding by the PfEMP1 family implicated in severe childhood malaria.

Cell Host Microbe 2015 Jan 4;17(1):118-29. Epub 2014 Dec 4.

Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU Oxford, UK. Electronic address:

The PfEMP1 family of surface proteins is central for Plasmodium falciparum virulence and must retain the ability to bind to host receptors while also diversifying to aid immune evasion. The interaction between CIDRα1 domains of PfEMP1 and endothelial protein C receptor (EPCR) is associated with severe childhood malaria. We combine crystal structures of CIDRα1:EPCR complexes with analysis of 885 CIDRα1 sequences, showing that the EPCR-binding surfaces of CIDRα1 domains are conserved in shape and bonding potential, despite dramatic sequence diversity. Additionally, these domains mimic features of the natural EPCR ligand and can block this ligand interaction. Using peptides corresponding to the EPCR-binding region, antibodies can be purified from individuals in malaria-endemic regions that block EPCR binding of diverse CIDRα1 variants. This highlights the extent to which such a surface protein family can diversify while maintaining ligand-binding capacity and identifies features that should be mimicked in immunogens to prevent EPCR binding.
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http://dx.doi.org/10.1016/j.chom.2014.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297295PMC
January 2015

Cellular manganese content is developmentally regulated in human dopaminergic neurons.

Sci Rep 2014 Oct 28;4:6801. Epub 2014 Oct 28.

1] Department of Neurology, Vanderbilt University Medical Center, Nashville, TN [2] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN.

Manganese (Mn) is both an essential biological cofactor and neurotoxicant. Disruption of Mn biology in the basal ganglia has been implicated in the pathogenesis of neurodegenerative disorders, such as parkinsonism and Huntington's disease. Handling of other essential metals (e.g. iron and zinc) occurs via complex intracellular signaling networks that link metal detection and transport systems. However, beyond several non-selective transporters, little is known about the intracellular processes regulating neuronal Mn homeostasis. We hypothesized that small molecules that modulate intracellular Mn could provide insight into cell-level Mn regulatory mechanisms. We performed a high throughput screen of 40,167 small molecules for modifiers of cellular Mn content in a mouse striatal neuron cell line. Following stringent validation assays and chemical informatics, we obtained a chemical 'toolbox' of 41 small molecules with diverse structure-activity relationships that can alter intracellular Mn levels under biologically relevant Mn exposures. We utilized this toolbox to test for differential regulation of Mn handling in human floor-plate lineage dopaminergic neurons, a lineage especially vulnerable to environmental Mn exposure. We report differential Mn accumulation between developmental stages and stage-specific differences in the Mn-altering activity of individual small molecules. This work demonstrates cell-level regulation of Mn content across neuronal differentiation.
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http://dx.doi.org/10.1038/srep06801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210885PMC
October 2014

Discoidin domain receptor 1 (DDR1) kinase as target for structure-based drug discovery.

Drug Discov Today 2015 Feb 7;20(2):255-61. Epub 2014 Oct 7.

Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA. Electronic address:

Discoidin domain receptor (DDR) 1 and 2 are transmembrane receptors that belong to the family of receptor tyrosine kinases (RTK). Upon collagen binding, DDRs transduce cellular signaling involved in various cell functions, including cell adhesion, proliferation, differentiation, migration, and matrix homeostasis. Altered DDR function resulting from either mutations or overexpression has been implicated in several types of disease, including atherosclerosis, inflammation, cancer, and tissue fibrosis. Several established inhibitors, such as imatinib, dasatinib, and nilotinib, originally developed as Abelson murine leukemia (Abl) kinase inhibitors, have been found to inhibit DDR kinase activity. As we review here, recent discoveries of novel inhibitors and their co-crystal structure with the DDR1 kinase domain have made structure-based drug discovery for DDR1 amenable.
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http://dx.doi.org/10.1016/j.drudis.2014.09.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336622PMC
February 2015

Identification of NAD(P)H quinone oxidoreductase activity in azoreductases from P. aeruginosa: azoreductases and NAD(P)H quinone oxidoreductases belong to the same FMN-dependent superfamily of enzymes.

PLoS One 2014 10;9(6):e98551. Epub 2014 Jun 10.

Department of Pharmacology, University of Oxford, Oxford, United Kingdom; Faculty of Science, Engineering and Computing, Kingston University, Kingston upon Thames, United Kingdom.

Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098551PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051601PMC
October 2015

Computational methods in drug discovery.

Pharmacol Rev 2014 31;66(1):334-95. Epub 2013 Dec 31.

Jr., Center for Structural Biology, 465 21st Ave South, BIOSCI/MRBIII, Room 5144A, Nashville, TN 37232-8725.

Computer-aided drug discovery/design methods have played a major role in the development of therapeutically important small molecules for over three decades. These methods are broadly classified as either structure-based or ligand-based methods. Structure-based methods are in principle analogous to high-throughput screening in that both target and ligand structure information is imperative. Structure-based approaches include ligand docking, pharmacophore, and ligand design methods. The article discusses theory behind the most important methods and recent successful applications. Ligand-based methods use only ligand information for predicting activity depending on its similarity/dissimilarity to previously known active ligands. We review widely used ligand-based methods such as ligand-based pharmacophores, molecular descriptors, and quantitative structure-activity relationships. In addition, important tools such as target/ligand data bases, homology modeling, ligand fingerprint methods, etc., necessary for successful implementation of various computer-aided drug discovery/design methods in a drug discovery campaign are discussed. Finally, computational methods for toxicity prediction and optimization for favorable physiologic properties are discussed with successful examples from literature.
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http://dx.doi.org/10.1124/pr.112.007336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880464PMC
June 2014

Structure of arylamine N-acetyltransferase from Mycobacterium tuberculosis determined by cross-seeding with the homologous protein from M. marinum: triumph over adversity.

Acta Crystallogr D Biol Crystallogr 2013 Aug 17;69(Pt 8):1433-46. Epub 2013 Jul 17.

Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, England.

Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) plays an important role in the intracellular survival of the microorganism inside macrophages. Medicinal chemistry efforts to optimize inhibitors of the TBNAT enzyme have been hampered by the lack of a three-dimensional structure of the enzyme. In this paper, the first structure of TBNAT, determined using a lone crystal produced using cross-seeding with the homologous protein from M. marinum, is reported. Despite the similarity between the two enzymes (74% sequence identity), they show distinct physical and biochemical characteristics. The structure elegantly reveals the characteristic features of the protein surface as well as details of the active site of TBNAT relevant to drug-discovery efforts. The crystallographic analysis of the diffraction data presented many challenges, since the crystal was twinned and the habit possessed pseudo-translational symmetry.
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http://dx.doi.org/10.1107/S0907444913015126DOI Listing
August 2013

Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry.

Proc Natl Acad Sci U S A 2013 Jul 24;110(28):11373-8. Epub 2013 Jun 24.

Laboratory of Biomolecular Research (LBR), Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.

Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species.
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http://dx.doi.org/10.1073/pnas.1302721110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710844PMC
July 2013

Structural basis for duplex RNA recognition and cleavage by Archaeoglobus fulgidus C3PO.

Nat Struct Mol Biol 2013 Mar 27;20(3):380-6. Epub 2013 Jan 27.

Department of Biochemistry, University of Oxford, Oxford, UK.

Oligomeric complexes of Trax and Translin proteins, known as C3POs, participate in several eukaryotic nucleic acid metabolism pathways, including RNA interference and tRNA processing. In RNA interference in humans and Drosophila, C3PO activates the RNA-induced silencing complex (RISC) by removing the passenger strand of the small interfering RNA precursor duplex, using nuclease activity present in Trax. How C3POs engage with nucleic acid substrates is unknown. Here we identify a single protein from Archaeoglobus fulgidus that assembles into an octamer highly similar to human C3PO. The structure in complex with duplex RNA reveals that the octamer entirely encapsulates a single 13-base-pair RNA duplex inside a large inner cavity. Trax-like-subunit catalytic sites target opposite strands of the duplex for cleavage separated by 7 base pairs. The structure provides insight into the mechanism of RNA recognition and cleavage by an archaeal C3PO-like complex.
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http://dx.doi.org/10.1038/nsmb.2487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597040PMC
March 2013

Benchmarking ligand-based virtual High-Throughput Screening with the PubChem database.

Molecules 2013 Jan 8;18(1):735-56. Epub 2013 Jan 8.

Department of Chemistry, Pharmacology, and Biomedical Informatics, Center for Structural Biology, Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA.

With the rapidly increasing availability of High-Throughput Screening (HTS) data in the public domain, such as the PubChem database, methods for ligand-based computer-aided drug discovery (LB-CADD) have the potential to accelerate and reduce the cost of probe development and drug discovery efforts in academia. We assemble nine data sets from realistic HTS campaigns representing major families of drug target proteins for benchmarking LB-CADD methods. Each data set is public domain through PubChem and carefully collated through confirmation screens validating active compounds. These data sets provide the foundation for benchmarking a new cheminformatics framework BCL::ChemInfo, which is freely available for non-commercial use. Quantitative structure activity relationship (QSAR) models are built using Artificial Neural Networks (ANNs), Support Vector Machines (SVMs), Decision Trees (DTs), and Kohonen networks (KNs). Problem-specific descriptor optimization protocols are assessed including Sequential Feature Forward Selection (SFFS) and various information content measures. Measures of predictive power and confidence are evaluated through cross-validation, and a consensus prediction scheme is tested that combines orthogonal machine learning algorithms into a single predictor. Enrichments ranging from 15 to 101 for a TPR cutoff of 25% are observed.
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http://dx.doi.org/10.3390/molecules18010735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759399PMC
January 2013
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