Crystallographic approach to fragment-based hit discovery against Schistosoma mansoni purine nucleoside phosphorylase.

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

Patient Engagement: an Assessment of Canadian Radiotherapy Programs' Current Practices, Perceived Barriers, and Facilitators.

Patient engagement and education have been mandated across Canadian radiation oncology programs (ROP). Guidance documents include the 2014 Canadian Association of Radiation Oncology (CARO) Radiation Therapy Patient Charter, the 2016 Canadian Partnership for Quality Radiotherapy (CPQR) Patient Engagement Guidelines (PEG) for Canadian Radiation Treatment Programs, and Accreditation Canada's 2017 refresh of Cancer Care Standards. Since little is known regarding uptake of these guidance statements, Canadian ROP were surveyed to assess current patient engagement and education practices. An e-survey was sent to Canadian ROP (n = 44). The survey focused on awareness and uptake of the CARO Patient Charter, CPQR PEG, and patient education practices. Survey development was guided by these documents and expert consensus, including CARO's Quality and Standards Patient Education/Engagement working group. Many (71%) responding ROP were familiar with the CARO Patient Charter, while 24% reported use. More than half (53%) of ROP were aware of the CPQR PEG, but approximately third (37%) had previously completed a self-audit. Most (88%) ROP view a pan-Canadian, evidence-based approach to educational materials beneficial and feasible (80%), with the majority (89%) willing to share their best practices across the radiotherapy community. Patient engagement and education are nationally mandated and supported by guidance documents. However, gaps have been identified across ROP for awareness and use of available tools, as well as uptake of their processes critical to quality of care. Understanding current practices will inform CPQR/CARO-supported pan-Canadian initiatives to optimize uptake, including development of CPQR Patient Education Guidance for Canadian Radiation Treatment Programs.

Structure of a collagen VI α3 chain VWA domain array: adaptability and functional implications of myopathy causing mutations.

Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein-protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.

The Dual PDZ Domain from Postsynaptic Density Protein 95 Forms a Scaffold with Peptide Ligand.

PSD-95 is a member of the membrane-associated guanylate kinase class of proteins that forms scaffolding interactions with partner proteins, including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/disks large/zona occludens (PDZ) domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of this dual domain in both apo- and ligand-bound form: thermodynamic analysis of the ligand association and small-angle x-ray scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a three-dimensional scaffold that can be described by a space group. The concentration of the components in this study is comparable with those found in compartments of excitable cells such as the postsynaptic density and juxtaparanodes of Ranvier. These in vitro experiments inform the basis of the scaffolding function of PSD-95 and provide a detailed model for scaffold formation by the PDZ domains of PSD-95.

Functional and phylogenetic evidence of a bacterial origin for the first enzyme in sphingolipid biosynthesis in a phylum of eukaryotic protozoan parasites.

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause fetal damage and abortion in both animals and humans. Sphingolipids are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Here we report the identification, isolation, and analyses of the Toxoplasma serine palmitoyltransferase, an enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA. In all eukaryotes analyzed to date, serine palmitoyltransferase is a highly conserved heterodimeric enzyme complex. However, biochemical and structural analyses demonstrated the apicomplexan orthologue to be a functional, homodimeric serine palmitoyltransferase localized to the endoplasmic reticulum. Furthermore, phylogenetic studies indicated that it was evolutionarily related to the prokaryotic serine palmitoyltransferase, identified in the Sphingomonadaceae as a soluble homodimeric enzyme. Therefore this enzyme, conserved throughout the Apicomplexa, is likely to have been obtained via lateral gene transfer from a prokaryote.

Surveying GPCR solubilisation conditions using surface plasmon resonance.

Biophysical screening techniques, such as surface plasmon resonance, enable detailed kinetic analysis of ligands binding to solubilised G-protein coupled receptors. The activity of a receptor solubilised out of the membrane is crucially dependent on the environment in which it is suspended. Finding the right conditions is challenging due to the number of variables to investigate in order to determine the optimum solubilisation buffer for any given receptor. In this study we used surface plasmon resonance technology to screen a variety of solubilisation conditions including buffers and detergents for two model receptors: CXCR4 and CCR5. We tested 950 different combinations of solubilisation conditions for both receptors. The activity of both receptors was monitored by using conformation dependent monoclonal antibodies and the binding of small molecule ligands. Despite both receptors belonging to the chemokine receptor family they show some differences in their preference for solubilisation conditions that provide the highest level of binding for both the conformation dependent antibodies and small molecules. The study described here is focused not only on finding the best solubilisation conditions for each receptor, but also on factors that determine the sensitivity of the assay for each receptor. We also suggest how these data about different buffers and detergents can be used as a guide for selecting solubilisation conditions for other membrane proteins.

SilE is an intrinsically disordered periplasmic "molecular sponge" involved in bacterial silver resistance.

Ag(+) resistance was initially found on the Salmonella enetrica serovar Typhimurium multi-resistance plasmid pMG101 from burns patients in 1975. The putative model of Ag(+) resistance, encoded by the sil operon from pMG101, involves export of Ag(+) via an ATPase (SilP), an effluxer complex (SilCFBA) and a periplasmic chaperon of Ag(+) (SilE). SilE is predicted to be intrinsically disordered. We tested this hypothesis using structural and biophysical studies and show that SilE is an intrinsically disordered protein in its free apo-form but folds to a compact structure upon optimal binding to six Ag(+) ions in its holo-form. Sequence analyses and site-directed mutagenesis established the importance of histidine and methionine containing motifs for Ag(+) -binding, and identified a nucleation core that initiates Ag(+) -mediated folding of SilE. We conclude that SilE is a molecular sponge for absorbing metal ions.

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

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.

Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters.

The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years.

Codon optimization and factorial screening for enhanced soluble expression of human ciliary neurotrophic factor in Escherichia coli.

BACKGROUND: Neurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system. Human ciliary neurotrophic factor (hCNTF) has neuroprotective properties and is also known to influence energy balance. Consequently, hCNTF has potential therapeutic applications in neurodegenerative, obesity and diabetes related disorders. Clinical and biological applications of hCNTF necessitate a recombinant expression system to produce large amounts of functional protein in soluble form. Earlier attempts to express hCNTF in Escherichia coli (E. coli) were limited by low amounts and the need to refold from inclusion bodies. RESULTS: In this report, we describe a strategy to effectively identify constructs and conditions for soluble expression of hCNTF in E. coli. Small-scale expression screening with soluble fusion tags identified many conditions that yielded soluble expression. Codon optimized 6-His-hCNTF construct showed soluble expression in all the conditions tested. Large-scale culture of the 6-His-hCNTF construct yielded high (10 - 20 fold) soluble expression (8 - 9 fold) as compared to earlier published reports. Functional activity of recombinant 6-His-hCNTF produced was confirmed by its binding to hCNTF receptor (hCNTFRα) with an EC50 = 36 nM. CONCLUSION: Our results highlight the combination of codon optimization and screening soluble fusion tags as a successful strategy for high yielding soluble expression of hCNTF in E. coli. Codon optimization of the hCNTF sequence seems to be sufficient for soluble expression of hCNTF. The combined approach of codon optimization and soluble fusion tag screen can be an effective strategy for soluble expression of pharmaceutical proteins in E. coli.

Structure and assembly of a trans-periplasmic channel for type IV pili in Neisseria meningitidis.

Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer membrane is mediated by a specialized secretin protein, PilQ, but the way in which this large channel is formed is unknown. Using NMR, we derived the structures of the periplasmic domains from N. meningitidis PilQ: the N-terminus is shown to consist of two ß-domains, which are unique to the type IV pilus-dependent secretins. The structure of the second ß-domain revealed an eight-stranded ß-sandwich structure which is a novel variant of the HSP20-like fold. The central part of PilQ consists of two α/ß fold domains: the structure of the first of these is similar to domains from other secretins, but with an additional α-helix which links it to the second α/ß domain. We also determined the structure of the entire PilQ dodecamer by cryoelectron microscopy: it forms a cage-like structure, enclosing a cavity which is approximately 55 Å in internal diameter at its largest extent. Specific regions were identified in the density map which corresponded to the individual PilQ domains: this allowed us to dock them into the cryoelectron microscopy density map, and hence reconstruct the entire PilQ assembly which spans the periplasm. We also show that the C-terminal domain from the lipoprotein PilP, which is essential for pilus assembly, binds specifically to the first α/ß domain in PilQ and use NMR chemical shift mapping to generate a model for the PilP:PilQ complex. We conclude that passage of the pilus fiber requires disassembly of both the membrane-spanning and the ß-domain regions in PilQ, and that PilP plays an important role in stabilising the PilQ assembly during secretion, through its anchorage in the inner membrane.

Structural and functional characterization of the kindlin-1 pleckstrin homology domain.

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin ß-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P(3); K(D) ∼100 µM) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P(3) as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P(2) on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P(3) is affected by the presence of PO(4) ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.

S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA.

Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo(5)U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo(5)U and was annotated as an S-adenosyl-L-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-L-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxymethyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-ray structure, PDB entry 1im8, suggests that the active site contains SCM-SAH and not SAM.

Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1{alpha} localization in erythroblasts.

Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material by Western blot. A detailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genome-wide epigenetic landscape of several histone modifications is maintained. However, immunofluorescence analysis of intermediate erythroblasts from patients with CDA-1 reveals abnormal accumulation of HP1α in the Golgi apparatus. A link between mutant codanin-1 and the aberrant localization of HP1α is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1α antibodies. Furthermore, we show colocalization of codanin-1 with Sec23B, the protein defective in CDA-2 suggesting that the CDAs might be linked at the molecular level. Mice containing a gene-trapped Cdan1 locus demonstrate its widespread expression during development. Cdan1(gt/gt) homozygotes die in utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis.

Application of particle swarm optimization to understand the mechanism of action of allosteric inhibitors of the enzyme HSD17ß13.

Understanding a drug candidate's mechanism of action is crucial for its further development. However, kinetic schemes are often complex and multi-parametric, especially for proteins in oligomerization equilibria. Here, we demonstrate the use of particle swarm optimization (PSO) as a method to select between different sets of parameters that are too far apart in the parameter space to be found by conventional approaches. PSO is based upon the swarming of birds: each bird in the flock assesses multiple landing spots while at the same time sharing that information with its neighbors. We applied this approach to the kinetics of HSD17ß13 enzyme inhibitors, which displayed unusually large thermal shifts. Thermal shift data for HSD17ß13 indicated that the inhibitor shifted the oligomerization equilibrium toward the dimeric state. Validation of the PSO approach was provided by experimental mass photometry data. These results encourage further exploration of multi-parameter optimization algorithms as tools in drug discovery.

High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli.

A suite of protein fusion vectors is presented that has been designed so that nine separate fusion vectors can be constructed from one PCR product using InFusion™ cloning. These vectors in combination with a small scale Escherichia coli expression screen can be used to assess in parallel the effect of fusion tags on solubility. The vectors were tested with 20 target proteins and the results suggest that the vectors are useful both as a rescue strategy if the N-terminal hexa-histidine tagged construct does not express and also as part of a primary expression experiment.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Šresolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and ß D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

Striving to Fill in Gaps between Clinical Practice and Standards: The Evolution of a Pan-Canadian Approach to Patient-Reported Outcomes Use.

Despite the known importance and necessity of the standardized collection and use of patient-reported outcomes (PROs), there remain challenges to successful clinical implementation. Facilitated through a quality improvement initiative spearheaded by the Canadian Partnership for Quality Radiotherapy (CPQR), and now guided by the Canadian Association of Radiation Oncology (CARO)'s Quality and Standards Committee, patient representatives and early-adopter radiation treatment programs continue to champion the expansion of PROs initiatives across the country. The current review discusses the evolution of a pan-Canadian approach to PROs use, striving to fill in gaps between clinical practice and guideline recommendations through multi-centre and multidisciplinary collaboration.

Surface Plasmon Resonance Screening to Identify Active and Selective Adenosine Receptor Binding Fragments.

Surface plasmon resonance (SPR) is a widely used method to study ligand-protein interactions. The throughput and sensitivity of SPR has made it an important technology for measuring low-affinity, ultralow weight fragments (<200 Da) in the early stages of drug discovery. However, the biochemistry of membrane proteins, such as G-protein-coupled receptors (GPCRs), makes their SPR fragment screening particularly challenging, especially for native/wild-type, nonthermostabilized mutant receptors. In this study, we demonstrate the use of SPR-based biosensors to study the entire human family of adenosine receptors and present biologically active novel binders with a range of selectivity to human adenosine 2a receptor (hA2AR) from an ultralow weight fragment library and the public GlaxoSmithKline (GSK) kinase library. Thus, we demonstrate the ability of SPR to screen ultra-low-affinity fragments and identify biologically meaningful chemical equity and that SPR campaigns are highly effective "chemical filters" for screening small building block fragments that can be used to enable drug discovery programs.