The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity.

A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Å resolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce.

Cyclophilins (CYPs) are a group of ubiquitous prolyl cis/trans isomerases (PPIases). It was shown that plants possess the most diverse CYP families and that these are abundant in the phloem long-distance translocation stream. Since phloem exudate showed PPIase activity, three single-domain CYPs that occur in phloem samples from Brassica napus were characterised on functional and structural levels. It could be shown that they exhibit isomerase activity and that this activity is controlled by a redox regulation mechanism, which has been postulated for divergent CYPs. The structure determination by small-angle X-ray scattering experiments revealed a conserved globular shape. In addition, the high-resolution crystal structure of BnCYP19-1 was resolved and refined to 2.0 Å resolution, and the active sites of related CYPs as well as substrate binding were modelled. The obtained data and results support the hypothesis that single domain phloem CYPs are active phloem PPIases that may function as chaperones.

Humanity is facing an increasing health threat caused by a variety of multidrug resistant bacteria. Within this scenario, Staphylococcus aureus, in particular methicillin resistant S. aureus (MRSA), is responsible for a number of hospital-acquired bacterial infections. The emergence of microbial antibiotic resistance urgently requires the identification of new and innovative strategies to treat antibiotic resistant microorganisms. In this context, structure and function analysis of potential drug targets in metabolic pathways vital for bacteria endurance, such as the vitamin K2 synthesis pathway, becomes interesting. We have solved and refined the crystal structure of the S. aureus DHNA thioesterase (SaDHNA), a key enzyme in the vitamin K2 pathway. The crystallographic structure in combination with small angle X-ray solution scattering data revealed a functional tetramer of SaDHNA. Complementary activity assays of SaDHNA indicated a preference for hydrolysing long acyl chains. Site-directed mutagenesis of SaDHNA confirmed the functional importance of Asp16 and Glu31 for thioesterase activity and substrate binding at the putative active site, respectively. Docking studies were performed and rational designed peptides were synthesized and tested for SaDHNA inhibition activity. The high-resolution structure of SaDHNA and complementary information about substrate binding will support future drug discovery and design investigations to inhibit the vitamin K2 synthesis pathway.

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physio-logical temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system - the CFEL TapeDrive - with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.

Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin). Thiamin in its activated form, thiamin pyrophosphate, is an essential co-factor for all organisms. Therefore, thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional co-factors by the bacterium can function as pro-drugs which thus block various co-factor dependent pathways. We characterized one of the key enzymes within the S. aureus vitamin B1 biosynthetic pathway, 5-(hydroxyethyl)-4-methylthiazole kinase (SaThiM; EC 2.7.1.50), a potential target for pro-drug compounds and analyzed the native structure of SaThiM and complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues.

Plasmodium species are protozoan parasites causing the deadly malaria disease. They have developed effective resistance mechanisms against most antimalarial medication, causing an urgent need to identify new antimalarial drug targets. Ideally, new drugs would be generated to specifically target the parasite with minimal or no toxicity to humans, requiring these drug targets to be distinctly different from the host's metabolic processes or even absent in the host. In this context, the essential presence of vitamin B6 biosynthesis enzymes in Plasmodium, the pyridoxal phosphate (PLP) biosynthesis enzyme complex, and its absence in humans is recognized as a potential drug target. To characterize the PLP enzyme complex in terms of initial drug discovery investigations, we performed structural analysis of the Plasmodium vivax PLP synthase domain (Pdx1), glutaminase domain (Pdx2), and Pdx1-Pdx2 (Pdx) complex (PLP synthase complex) by utilizing complementary bioanalytical techniques, such as dynamic light scattering (DLS), X-ray solution scattering (SAXS), and electron microscopy (EM). Our investigations revealed a dodecameric Pdx1 and a monodispersed Pdx complex. Pdx2 was identified in monomeric and in different oligomeric states in solution. Interestingly, mixing oligomeric and polydisperse Pdx2 with dodecameric monodisperse Pdx1 resulted in a monodispersed Pdx complex. SAXS measurements revealed the low-resolution dodecameric structure of Pdx1, different oligomeric structures for Pdx2, and a ring-shaped dodecameric Pdx1 decorated with Pdx2, forming a heteromeric 24-meric Pdx complex.

Although it is normally an innocuous part of the human skin microbiota, Staphylococcus epidermidis has emerged as a major nosocomial pathogen, and implanted foreign materials are an essential risk factor for the development of an infection. The extraordinary efficiency of S. epidermidis to colonize artificial surfaces is particularly related to the ability to form biofilms. Biofilm formation itself critically depends on stable pathogen binding to extracellular host matrix components, e.g. fibronectin (Fn), covering inserted devices in vast amounts. Extracellular matrix binding protein (Embp) and its subdomains referred to as the F-repeat and the FG-repeat are critical for adherence of S. epidermidis to surface-immobilized Fn. Embp-Fn interactions preferentially occur with surface-bound, but not folded, globular Fn via binding to the F3 domain. High-resolution structure analysis of F- and FG-repeats revealed that both repeats are composed of two tightly connected triple α-helix bundles, exhibiting an elongated but rather rigid structural organization in solution. Both F- and FG-repeat possess Fn-binding capacity via interactions with type III subdomain FN12, involving residues within the C and F β-sheet. FN12 essentially supports stability of the globular Fn state, and thus these findings reasonably explain why Embp-mediated interaction of S. epidermidis necessitates Fn surface immobilization. Thus, Embp employs an uncharacterized bacterial Fn-binding mechanism to promote staphylococcal adherence.IMPORTANCEStaphylococcus epidermidis is a leading pathogen in implant-associated hospital infections. The pathogenesis critically depends on bacterial binding to ECM components, specifically fibronectin (Fn). The cell surface-localized, 1-MDa extracellular matrix binding protein (Embp) is essentially characterized by 10 F- and 40 FG-repeats. These repetitive units, each characterized by two α-helical bundles, organize themselves in a rigid, elongated form. Embp binds preferentially to surface-localized but not soluble Fn, with both F- and FG-repeats being sufficient for Fn binding and resulting bacterial adherence. Binding preferentially involves Fn type III domain, specifically residues of FN12 β-sheets C and F. Both play key role in stabilizing the globular Fn conformation, explaining the necessity of Fn surface immobilization for a subsequent interaction with Embp. In comparison to many other bacterial Fn-binding proteins using the Fn N terminus, Embp employs a previously undescribed mechanism supporting the adhesion of S. epidermidis to surface-immobilized Fn.

Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate-like density for nuclear-envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations.

The notoriety of parasitic nematode survival is directly related to chronic pathogenicity, which is evident in human lymphatic filariasis. It is a disease of poverty which causes severe disability affecting more than 120 million people worldwide. These nematodes down-regulate host immune system through a myriad of strategies that includes secretion of antioxidant and detoxification enzymes like glutathione-S-transferases (GSTs). Earlier studies have shown Wuchereria bancrofti GST to be a potential therapeutic target. Parasite GSTs catalyse the conjugation of glutathione to xenobiotic and other endogenous electrophiles and are essential for their long-term survival in lymph tissues. Hence, the crystal structure of WbGST along with its cofactor GSH at 2.3 Å resolution was determined. Structural comparisons against host GST reveal distinct differences in the substrate binding sites. The parasite xenobiotic binding site is more substrate/solvent accessible. The structure also suggests the presence of putative non-catalytic binding sites that may permit sequestration of endogenous and exogenous ligands. The structure of WbGST also provides a case for the role of the π-cation interaction in stabilizing catalytic Tyr compared to stabilization interactions described for other GSTs. Hence, the obtained information regarding crucial differences in the active sites will support future design of parasite specific inhibitors. Further, the study also evaluates the inhibition of WbGST and its variants by antifilarial diethylcarbamazine through kinetic assays.

Staphylotrichum longicolleum FW57 (DSM105789) is a prolific chitinolytic fungus isolated from wood, with a chitinase activity of 0.11 ± 0.01 U/mg. We selected this strain for genome sequencing and annotation, and compiled its growth characteristics on four different chitinous substrates as well as two agro-industrial waste products. We found that the enzymatic mixture secreted by FW57 was not only able to digest pre-treated sugarcane bagasse, but also untreated sugarcane bagasse and maize leaves. The efficiency was comparable to a commercial enzymatic cocktail, highlighting the potential of the S. longicolleum enzyme mixture as an alternative pretreatment method. To further characterize the enzymes, which efficiently digested polymers such as cellulose, hemicellulose, pectin, starch, and lignin, we performed in-depth mass spectrometry-based secretome analysis using tryptic peptides from in-gel and in-solution digestions. Depending on the growth conditions, we were able to detect from 442 to 1092 proteins, which were annotated to identify from 134 to 224 putative carbohydrate-active enzymes (CAZymes) in five different families: glycoside hydrolases, auxiliary activities, carbohydrate esterases, polysaccharide lyases, glycosyl transferases, and proteins containing a carbohydrate-binding module, as well as combinations thereof. The FW57 enzyme mixture could be used to replace commercial enzyme cocktails for the digestion of agro-residual substrates.

Detergents are widely used for the isolation and solubilization of membrane proteins to support crystallization and structure determination. Detergents are amphiphilic molecules that form micelles once the characteristic critical micelle concentration (CMC) is achieved and can solubilize membrane proteins by the formation of micelles around them. The results are presented of a study of micelle formation observed by in situ dynamic light-scattering (DLS) analyses performed on selected detergent solutions using a newly designed advanced hardware device. DLS was initially applied in situ to detergent samples with a total volume of approximately 2 µl. When measured with DLS, pure detergents show a monodisperse radial distribution in water at concentrations exceeding the CMC. A series of all-trans n-alkyl-β-D-maltopyranosides, from n-hexyl to n-tetradecyl, were used in the investigations. The results obtained verify that the application of DLS in situ is capable of distinguishing differences in the hydrodynamic radii of micelles formed by detergents differing in length by only a single CH2 group in their aliphatic tails. Subsequently, DLS was applied to investigate the distribution of hydrodynamic radii of membrane proteins and selected water-insoluble proteins in presence of detergent micelles. The results confirm that stable protein-detergent complexes were prepared for (i) bacteriorhodopsin and (ii) FetA in complex with a ligand as examples of transmembrane proteins. A fusion of maltose-binding protein and the Duck hepatitis B virus X protein was added to this investigation as an example of a non-membrane-associated protein with low water solubility. The increased solubility of this protein in the presence of detergent could be monitored, as well as the progress of proteolytic cleavage to separate the fusion partners. This study demonstrates the potential of in situ DLS to optimize solutions of protein-detergent complexes for crystallization applications.

Proteins constituting the presynaptic machinery of vesicle release undergo substantial conformational changes during the process of exocytosis. While changes in the conformation make proteins vulnerable to aggregation and degradation, little is known about synaptic chaperones which counteract these processes. We show that the cell adhesion molecule CHL1 directly interacts with and regulates the activity of the synaptic chaperones Hsc70, CSP and alphaSGT. CHL1, Hsc70, CSP and alphaSGT form predominantly CHL1/Hsc70/alphaSGT and CHL1/CSP complexes in synapses. Among the various complexes formed by CHL1, Hsc70, CSP and alphaSGT, SNAP25 and VAMP2 induce chaperone activity only in CHL1/Hsc70/alphaSGT and CHL1/CSP complexes, respectively, indicating a remarkable selectivity of a presynaptic chaperone activity for proteins of the exocytotic machinery. In mice with genetic ablation of CHL1, chaperone activity in synapses is reduced and the machinery for synaptic vesicle exocytosis and, in particular, the SNARE complex is unable to sustain prolonged synaptic activity. Thus, we reveal a novel role for a cell adhesion molecule in selective activation of the presynaptic chaperone machinery.

Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm(3), a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals.

The transition to a biobased economy involving the depolymerization and fermentation of renewable agro-industrial sources is a challenge that can only be met by achieving the efficient hydrolysis of biomass to monosaccharides. In nature, lignocellulosic biomass is mainly decomposed by fungi. We recently identified six efficient cellulose degraders by screening fungi from Vietnam.

To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.

The gene coding for the aminoglycoside adenylyltransferase (aadA6) from a clinical isolate of Pseudomonas aeruginosa was cloned and expressed in Escherichia coli strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5 mM MgCl2 for normal Michaelis-Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1 mM MgCl2. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100 μM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin.

Latest advancement of omics technologies allows in-depth characterization of venom compositions. In the present work we present a proteomic study of two snake venoms of the genus Naja i.e., Naja naja (black cobra) and Naja oxiana (brown cobra) of Pakistani origin. The present study has shown that these snake venoms consist of a highly diversified proteome. Furthermore, the data also revealed variation among closely related species. High throughput mass spectrometric analysis of the venom proteome allowed to identify for the N. naja venom 34 protein families and for the N. oxiana 24 protein families. The comparative evaluation of the two venoms showed that N. naja consists of a more complex venom proteome than N. oxiana venom. Analysis also showed N-terminal acetylation (N-ace) of a few proteins in both venoms. To the best of our knowledge, this is the first study revealing this posttranslational modification in snake venom. N-ace can shed light on the mechanism of regulation of venom proteins inside the venom gland. Furthermore, our data showed the presence of other body proteins, e.g., ankyrin repeats, leucine repeats, zinc finger, cobra serum albumin, transferrin, insulin, deoxyribonuclease-2-alpha, and other regulatory proteins in these venoms. Interestingly, our data identified Ras-GTpase type of proteins, which indicate the presence of extracellular vesicles in the venom. The data can support the production of distinct and specific anti-venoms and also allow a better understanding of the envenomation and mechanism of distribution of toxins. Data are available via ProteomeXchange with identifier PXD018726.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Many biochemical processes take place on timescales ranging from femto-seconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach for in situ crystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime.