The structure of nontypeable Haemophilus influenzae SapA in a closed conformation reveals a constricted ligand-binding cavity and a novel RNA binding motif.

Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 µM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 µM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.

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.

Immunogenicity profiling of protein antigens from capsular group B Neisseria meningitidis.

Outer membrane vesicle (OMV)- based vaccines have been used to provide strain-specific protection against capsular group B Neisseria meningitidis infections, but the full breadth of the immune response against the components of the OMV has not been established. Sera from adults vaccinated with an OMV vaccine were used to screen 91 outer membrane proteins (OMPs) incorporated in an antigen microarray panel. Antigen-specific IgG levels were quantified pre-vaccination, and after 12 and 18 weeks. These results were compared with IgG levels from mice vaccinated with the same OMV vaccine. The repertoires of highly responding antigens in humans and mice overlapped, but were not identical. The highest responding antigens to human IgG comprised four integral OMPs (PorA, PorB, OpcA and PilQ), a protein which promotes the stability of PorA and PorB (RmpM) and two lipoproteins (BamC and GNA1162). These observations will assist in evaluating the role of minor antigen components within OMVs in providing protection against meningococcal infection. In addition, the relative dominance of responses to integral OMPs in humans emphasizes the importance of this subclass and points to the value of maintaining conformational epitopes from integral membrane proteins in vaccine formulations.

Characterization of a Schistosoma mansoni NDPK expressed in sexual and digestive organs.

Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.

In vitro and in vivo characterization of the multiple isoforms of Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferases.

Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the "de novo" purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.

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.

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.

Optimization of Membrane Protein Production Using Titratable Strains of E. coli.

The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.

Structural and kinetic analysis of Schistosoma mansoni Adenylosuccinate Lyase (SmADSL).

Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5'-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites are formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway.

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome.

A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a Kb of 1.8 × 105 M-1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space Rg of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.

The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity.

Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.

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.

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.

Substituted 2-(2-aminopyrimidin-4-yl)pyridine-4-carboxylates as potent inhibitors of JumonjiC domain-containing histone demethylases.

BACKGROUND: Aberrant expression of iron(II)- and 2-oxoglutarate-dependent JumonjiC histone demethylases has been linked to cancer. Potent demethylase inhibitors are drug candidates and biochemical tools to elucidate the functional impact of demethylase inhibition. METHODS & RESULTS: Virtual screening identified a novel lead scaffold against JMJD2A with low-micromolar potency in vitro. Analogs were acquired from commercial sources respectively synthesized in feedback with biological testing. Optimized compounds were transformed into cell-permeable prodrugs. A cocrystal x-ray structure revealed the mode of binding of these compounds as competitive to 2-oxoglutarate and confirmed kinetic experiments. Selectivity studies revealed a preference for JMJD2A and JARID1A over JMJD3. CONCLUSION: Virtual screening and rational structural optimization led to a novel scaffold for highly potent and selective JMJD2A inhibitors.

Diversity between mammalian tolloid proteinases: Oligomerisation and non-catalytic domains influence activity and specificity.

The mammalian tolloid family of metalloproteinases is essential for tissue patterning and extracellular matrix assembly. The four members of the family: bone morphogenetic protein-1 (BMP-1), mammalian tolloid (mTLD), tolloid-like (TLL)-1 and TLL-2 differ in their substrate specificity and activity levels, despite sharing similar domain organization. We have previously described a model of substrate exclusion by dimerisation to explain differences in the activities of monomeric BMP-1 and dimers of mTLD and TLL-1. Here we show that TLL-2, the least active member of the tolloid family, is predominantly monomeric in solution, therefore it appears unlikely that substrate exclusion via dimerisation is a mechanism for regulating TLL-2 activity. X-ray scattering and electron microscopy structural and biophysical analyses reveal an elongated shape for the monomer and flexibility in the absence of calcium. Furthermore, we show that TLL-2 can cleave chordin in vitro, similar to other mammalian tolloids, but truncated forms of TLL-2 mimicking BMP-1 are unable to cleave chordin. However, both the N- and C-terminal non-catalytic domains from all mammalian tolloids bind chordin with high affinity. The mechanisms underlying substrate specificity and activity in the tolloid family are complex with variation between family members and depend on both multimerisation and substrate interaction.

Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport.

Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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.

Application of In-Fusion™ cloning for the parallel construction of E. coli expression vectors.

In-Fusion™ cloning is a flexible DNA ligase-independent cloning technology that has wide-ranging uses in molecular biology. In this chapter we describe the protocols used in the OPPF-UK to design and construct expression vectors using In-Fusion™. Our method for small scale expression screening in Escherichia coli of constructs generated by In-Fusion™ is also outlined.

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.