Role of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 in Inflammation and Pathogen-Associated Interactions.

BACKGROUND: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is known as a major receptor for oxidized low-density lipoproteins (oxLDL) and plays a significant role in the genesis of atherosclerosis. Recent research has shown its involvement in cancer, ischemic stroke, and diabetes. LOX-1 is a C-type lectin receptor and is involved in the activation of immune cells and inflammatory processes. It may further interact with pathogens, suggesting a role in infections or the host's response. SUMMARY: This review compiles the current knowledge of potential implications of LOX-1 in inflammatory processes and in host-pathogen interactions with a particular emphasis on its regulatory role in immune responses. Also discussed are genomic and structural variations found in LOX-1 homologs across different species as well as potential involvements of LOX-1 in inflammatory processes from the angle of different cell types and organ-specific interactions. KEY MESSAGES: The results presented reveal both similar and different structures in human and murine LOX-1 and provide clues as to the possible origins of different modes of interaction. These descriptions raise concerns about the suitability, particularly of mouse models, that are often used in the analysis of its functionality in humans. Further research should also aim to better understand the mostly unknown binding and interaction mechanisms between LOX-1 and different pathogens. This pursuit will not only enhance our understanding of LOX-1 involvement in inflammatory processes but also identify potential targets for immunomodulatory approaches.

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-ß Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

Towards Translation of PqsR Inverse Agonists: From In Vitro Efficacy Optimization to In Vivo Proof-of-Principle.

Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, which is involved in a wide range of dangerous infections. It develops alarming resistances toward antibiotic treatment. Therefore, alternative strategies, which suppress pathogenicity or synergize with antibiotic treatments are in great need to combat these infections more effectively. One promising approach is to disarm the bacteria by interfering with their quorum sensing (QS) system, which regulates the release of various virulence factors as well as biofilm formation. Herein, this work reports the rational design, optimization, and in-depth profiling of a new class of Pseudomonas quinolone signaling receptor (PqsR) inverse agonists. The resulting frontrunner compound features a pyrimidine-based scaffold, high in vitro and in vivo efficacy, favorable pharmacokinetics as well as clean safety pharmacology characteristics, which provide the basis for potential pulmonary as well as systemic routes of administration. An X-ray crystal structure in complex with PqsR facilitated further structure-guided lead optimization. The compound demonstrates potent pyocyanin suppression, synergizes with aminoglycoside antibiotic tobramycin against PA biofilms, and is active against a panel of clinical isolates from bronchiectasis patients. Importantly, this in vitro effect translated into in vivo efficacy in a neutropenic thigh infection model in mice providing a proof-of-principle for adjunctive treatment scenarios.

Moonlighting chaperone activity of the enzyme PqsE contributes to RhlR-controlled virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa, with the rhl circuit playing the leading role in late and chronic infections. The majority of traits controlled by rhl transcription factor RhlR depend on PqsE, a dispensable thioesterase in Pseudomonas Quinolone Signal (PQS) biosynthesis that interferes with RhlR through an enigmatic mechanism likely involving direct interaction of both proteins. Here we show that PqsE and RhlR form a 2:2 protein complex that, together with RhlR agonist N-butanoyl-L-homoserine lactone (C4-HSL), solubilizes RhlR and thereby renders the otherwise insoluble transcription factor active. We determine crystal structures of the complex and identify residues essential for the interaction. To corroborate the chaperone-like activity of PqsE, we design stability-optimized variants of RhlR that bypass the need for C4-HSL and PqsE in activating PqsE/RhlR-controlled processes of P. aeruginosa. Together, our data provide insight into the unique regulatory role of PqsE and lay groundwork for developing new P. aeruginosa-specific pharmaceuticals.

Divergent synthesis and biological evaluation of 2-(trifluoromethyl)pyridines as virulence-attenuating inverse agonists targeting PqsR.

A short and divergent route towards new derivatives of 2-(trifluoromethyl)pyridines as potent inverse agonists of the bacterial target PqsR against Pseudomonas aeruginosa (PA) infections is described. This Gram-negative pathogen causes severe nosocomial infections and common antibiotic treatment options are rendered ineffective due to resistance issues. Based on an earlier identified optimized hit, we conducted derivatization and rigidification attempts employing two central building blocks. The western part of the molecule is built up via a 2-(trifluoromethyl)pyridine head group equipped with a terminal alkyne. The eastern section is then introduced through aryliode motifs exploiting Sonogashira as well as Suzuki-type chemistry. Subsequent modification provided quick access to an array of compounds, allowed for deep SAR insights, and enabled to optimize the hit scaffold into a lead structure of nanomolar potency combined with favorable in vitro ADME/T features.

A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) - a crucial transcriptional regulator serving major functions in PA virulence - can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry-driven hit-to-lead optimization and in-depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter-gene with IC50 values as low as 200 and 11 × 10-9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI-tobramycin (Tob) combination against PA biofilms using a tailor-made squalene-derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32-fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker-mediated therapy against PA infections opening up avenues for preclinical development.

Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.

The expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.

The N-terminal peptide of the transglutaminase-activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross-linking sites.

Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin-A (SGPA), EC3.4.21.80; streptogrisin-B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC3.4.21.4. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).

Flexible Fragment Growing Boosts Potency of Quorum-Sensing Inhibitors against Pseudomonas aeruginosa Virulence.

Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-aminopyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa. We pursue an innovative treatment strategy by interfering with the Pseudomonas quinolone signal (PQS) quorum sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency.

Structure of a glutamine donor mimicking inhibitory peptide shaped by the catalytic cleft of microbial transglutaminase.

The protein cross-linking enzyme transglutaminase from Streptomyces mobaraensis (MTG) is frequently used to modify therapeutic proteins. In order to reveal the binding mode of glutamine donor substrates, we have now crystallized MTG covalently linked to large inhibitory peptides. A series of peptide structures were examined but DIPIGSKMTG, which was chloroacetylated at serine, was the only inhibitory molecule that resulted in an interpretable density map. We found that, besides the warhead (modified Ser6), Ile4 and Gly5 of the inhibitory peptide occupy the tight but extended hydrophobic bottom of the MTG-binding cleft. Both termini of the peptide protrude along the cleft walls almost perpendicular to the bottom of the extended cleft. This peptide model suggests a zipper-like cross-linking mechanism of self-assembled substrate proteins by MTG.

Destructive twisting of neutral metalloproteases: the catalysis mechanism of the Dispase autolysis-inducing protein from Streptomyces mobaraensis DSM 40487.

The Dispase autolysis-inducing protein (DAIP) is produced by Streptomyces mobaraensis to disarm neutral metalloproteases by decomposition. The absence of a catalytic protease domain led to the assumption that the seven-bladed ß-propeller protein DAIP causes structural modifications, thereby triggering autolysis. Determination of protein complexes consisting of DAIP and thermolysin or DAIP and a nonfunctional E138A bacillolysin variant supported this postulation. Protein twisting was indicated by DAIP-mediated inhibition of thermolysin while bacillolysin underwent immediate autolysis under the same conditions. Interestingly, an increase in SYPRO orange fluorescence allowed tracking of the fast degradation process. Similarly rapid autolysis of thermolysin mediated by DAIP was only observed upon the addition of amphiphilic compounds, which probably amplify the induced structural changes. DAIP further caused degradation of FITC-labeled E138A bacillolysin by trypsin, as monitored by a linear decrease in fluorescence polarization. The kinetic model, calculated from the obtained data, suggested a three-step mechanism defined by (a) fast DAIP-metalloprotease complex formation, (b) slower DAIP-mediated protein twisting, and (c) fragmentation. These results were substantiated by crystallized DAIP attached to a C-terminal helix fragment of thermolysin. Structural superposition of the complex with thermolysin is indicative of a conformational change upon binding to DAIP. Importantly, the majority of metalloproteases, also including homologs from various pathogens, are highly conserved at the autolysis-prone peptide bonds, suggesting their susceptibility to DAIP-mediated decomposition, which may offer opportunities for pharmaceutical applications. DATABASES: The atomic coordinates and structure factors (PDB ID: 6FHP) have been deposited in the Protein Data Bank (http://www.pdb.org/). ENZYMES: Aureolysin, EC 3.4.24.29; bacillolysin (Dispase, Gentlyase), EC 3.4.24.28; lasB (elastase), EC 3.4.24.4; subtilisin, EC 3.4.21.62; thermolysin, EC 3.4.24.27; transglutaminase, EC 2.3.2.13; trypsin, EC 3.4.21.4; vibriolysin (hemagglutinin(HA)/protease), EC 3.4.24.25.

Design, development and application of whole-cell based antibiotic-specific biosensor.

Synthetic biology techniques hold great promise for optimising the production of natural products by microorganisms. However, evaluating the phenotype of a modified bacterium represents a major bottleneck to the engineering cycle - particularly for antibiotic-producing actinobacteria strains, which grow slowly and are challenging to genetically manipulate. Here, we report the generation and application of antibiotic-specific whole-cell biosensor derived from TetR transcriptional repressor for use in identifying and optimising antibiotic producers. The constructed biosensor was successfully used to improve production of polyketide antibiotic pamamycin. However, an initial biosensor based on native genetic elements had inadequate dynamic and operating ranges. To overcome these limitations, we fine-tuned biosensor performance through alterations of the promoter and operator of output module and the ligand affinity of transcription factor module, which enabled us to deduce recommendations for building and application of actinobacterial biosensors.

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis.

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPIp ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPIp exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPIp accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPIp for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications.

Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed ß-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen.

Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal-cell biotechnology. Difficult-to-express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full-length protein constructs. Post-translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time-, labor-, and cost-intensive. It is clearly desirable to find an automated and miniaturized fast multi-sample screening method for protein expression in such systems. With this in mind, in this paper a high-throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide-binding Oligomerization Domain-containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid-based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. Biotechnol. Bioeng. 2016;113: 1975-1983. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site-Specific Conjugation.

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.

The crystal structure of siroheme decarboxylase in complex with iron-uroporphyrin III reveals two essential histidine residues.

The isobacteriochlorin heme d1 serves as an essential cofactor in the cytochrome cd1 nitrite reductase NirS that plays an important role for denitrification. During the biosynthesis of heme d1, the enzyme siroheme decarboxylase catalyzes the conversion of siroheme to 12,18-didecarboxysiroheme. This enzyme was discovered recently (Bali S, Lawrence AD, Lobo SA, Saraiva LM, Golding BT, Palmer DJ et al. Molecular hijacking of siroheme for the synthesis of heme and d1 heme. Proc Natl Acad Sci USA 2011;108:18260-5) and is only scarcely characterized. Here, we present the crystal structure of the siroheme decarboxylase from Hydrogenobacter thermophilus representing the first three-dimensional structure for this type of enzyme. The overall structure strikingly resembles those of transcriptional regulators of the Lrp/AsnC family. Moreover, the structure of the enzyme in complex with a substrate analog reveals first insights into its active-site architecture. Through site-directed mutagenesis and subsequent biochemical characterization of the enzyme variants, two conserved histidine residues within the active site are identified to be involved in substrate binding and catalysis. Based on our results, we propose a potential catalytic mechanism for the enzymatic reaction catalyzed by the siroheme decarboxylase.

In situ structural analysis of the Yersinia enterocolitica injectisome.

Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30-40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance. DOI:http://dx.doi.org/10.7554/eLife.00792.001.

Structural characterization of Spinacia oleracea trypsin inhibitor III (SOTI-III).

In recent decades, several canonical serine protease inhibitor families have been classified and characterized. In contrast to most trypsin inhibitors, those from garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) do not share sequence similarity and have been proposed to form the new Mirabilis serine protease inhibitor family. These 30-40-amino-acid inhibitors possess a defined disulfide-bridge topology and belong to the cystine-knot miniproteins (knottins). To date, no atomic structure of this inhibitor family has been solved. Here, the first structure of S. oleracea trypsin inhibitor III (SOTI-III), in complex with bovine pancreatic trypsin, is reported. The inhibitor was synthesized by solid-phase peptide synthesis on a multi-milligram scale and was assayed to test its inhibitory activity and binding properties. The structure confirmed the proposed cystine-bridge topology. The structural features of SOTI-III suggest that it belongs to a new canonical serine protease inhibitor family with promising properties for use in protein-engineering and medical applications.