A conserved strategy to attack collagen: The activator domain in bacterial collagenases unwinds triple-helical collagen.

Bacterial collagenases are important virulence factors, secreted by several pathogenic Clostridium, Bacillus, Spirochaetes, and Vibrio species. Yet, the mechanism by which these enzymes cleave collagen is not well understood. Based on biochemical and mutational studies we reveal that collagenase G (ColG) from Hathewaya histolytica recognizes and processes collagen substrates differently depending on their nature (fibrillar vs. soluble collagen); distinct dynamic interactions between the activator and peptidase domain are required based on the substrate type. Using biochemical and circular dichroism studies, we identify the presumed noncatalytic activator domain as the single-domain triple helicase that unwinds collagen locally, transiently, and reversibly.

Fragment-Based Design, Synthesis, and Characterization of Aminoisoindole-Derived Furin Inhibitors.

A 1H-isoindol-3-amine was identified as suitable P1 group for the proprotein convertase furin using a crystallographic screening with a set of 20 fragments known to occupy the S1 pocket of trypsin-like serine proteases. Its binding mode is very similar to that observed for the P1 group of benzamidine-derived peptidic furin inhibitors suggesting an aminomethyl substitution of this fragment to obtain a couplable P1 residue for the synthesis of substrate-analogue furin inhibitors. The obtained inhibitors possess a slightly improved picomolar inhibitory potency compared to their benzamidine-derived analogues. The crystal structures of two inhibitors in complex with furin revealed that the new P1 group is perfectly suited for incorporation in peptidic furin inhibitors. Selected inhibitors were tested for antiviral activity against respiratory syncytial virus (RSV) and a furin-dependent influenza A virus (SC35M/H7N7) in A549 human lung cells and demonstrated an efficient inhibition of virus activation and replication at low micromolar or even submicromolar concentrations. First results suggest that the Mas-related G-protein coupled receptor GPCR-X2 could be a potential off-target for certain benzamidine-derived furin inhibitors.

Arabidopsis thaliana Phytocystatin 6 Forms Functional Oligomer and Amyloid Fibril States.

Cystatins encode a high functional variability not only because of their ability to inhibit different classes of proteases but also because of their propensity to form oligomers and amyloid fibrils. Phytocystatins, essential regulators of protease activity in plants, specifically inhibit papain-like cysteine proteases (PLCPs) and legumains through two distinct cystatin domains. Mammalian cystatins can form amyloid fibrils; however, the potential for amyloid fibril formation of phytocystatins remains unknown. In this study, we demonstrate that Arabidopsis thaliana phytocystatin 6 (AtCYT6) exists as a mixture of monomeric, dimeric, and oligomeric forms in solution. Noncovalent oligomerization was facilitated by the N-terminal cystatin domain, while covalent dimerization occurred through disulfide bond formation in the interdomain linker. The noncovalent dimeric form of AtCYT6 retained activity against its target proteases, papain and legumain, albeit with reduced inhibitory potency. Additionally, we observed the formation of amyloid fibrils by AtCYT6 under acidic pH conditions and upon heating. The amyloidogenic potential could be attributed to the AtCYT6's N-terminal domain (AtCYT6-NTD). Importantly, AtCYT6 amyloid fibrils harbored inhibitory activities against both papain and legumain. These findings shed light on the oligomerization and amyloidogenic behavior of AtCYT6, expanding our understanding of phytocystatin biology and its potential functional implications for plant protease regulation.

Phytocystatin 6 is a context-dependent, tight-binding inhibitor of Arabidopsis thaliana legumain isoform ß.

Plant legumains are crucial for processing seed storage proteins and are critical regulators of plant programmed cell death. Although research on legumains boosted recently, little is known about their activity regulation. In our study, we used pull-down experiments to identify AtCYT6 as a natural inhibitor of legumain isoform ß (AtLEGß) in Arabidopsis thaliana. Biochemical analysis revealed that AtCYT6 inhibits both AtLEGß and papain-like cysteine proteases through two separate cystatin domains. The N-terminal domain inhibits papain-like proteases, while the C-terminal domain inhibits AtLEGß. Furthermore, we showed that AtCYT6 interacts with legumain in a substrate-like manner, facilitated by a conserved asparagine residue in its reactive center loop. Complex formation was additionally stabilized by charged exosite interactions, contributing to pH-dependent inhibition. Processing of AtCYT6 by AtLEGß suggests a context-specific regulatory mechanism with implications for plant physiology, development, and programmed cell death. These findings enhance our understanding of AtLEGß regulation and its broader physiological significance.

Quantitative cross-linking via engineered cysteines to study inter-domain interactions in bacterial collagenases.

Inter-domain movements act as important activity modulators in multi-domain proteins. Here, we present a protocol for inter-domain cross-linking via engineered cysteines. Using collagenase G (ColG) from Hathewaya histolytica as a model, we describe steps for the design, expression, purification, and cross-linking of the target protein. We detail a system to monitor the progress of the cross-linking reaction and to confirm the structural integrity of the purified cross-linked proteins. We anticipate this protocol to be readily adaptable to other multi-domain enzymes. For complete details on the use and execution of this protocol, please refer to Serwanja et al.1.

Legumain Functions as a Transient TrkB Sheddase.

While primarily found in endo-lysosomal compartments, the cysteine protease legumain can also translocate to the cell surface if stabilized by the interaction with the RGD-dependent integrin receptor αVß3. Previously, it has been shown that legumain expression is inversely related to BDNF-TrkB activity. Here we show that legumain can conversely act on TrkB-BDNF by processing the C-terminal linker region of the TrkB ectodomain in vitro. Importantly, when in complex with BDNF, TrkB was not cleaved by legumain. Legumain-processed TrkB was still able to bind BDNF, suggesting a potential scavenger function of soluble TrkB towards BDNF. The work thus presents another mechanistic link explaining the reciprocal TrkB signaling and δ-secretase activity of legumain, with relevance for neurodegeneration.

Inhibitors of the Elastase LasB for the Treatment of Pseudomonas aeruginosa Lung Infections.

Infections caused by the Gram-negative pathogen Pseudomonas aeruginosa are emerging worldwide as a major threat to human health. Conventional antibiotic monotherapy suffers from rapid resistance development, underlining urgent need for novel treatment concepts. Here, we report on a nontraditional approach to combat P. aeruginosa-derived infections by targeting its main virulence factor, the elastase LasB. We discovered a new chemical class of phosphonates with an outstanding in vitro ADMET and PK profile, auspicious activity both in vitro and in vivo. We established the mode of action through a cocrystal structure of our lead compound with LasB and in several in vitro and ex vivo models. The proof of concept of a combination of our pathoblocker with levofloxacin in a murine neutropenic lung infection model and the reduction of LasB protein levels in blood as a proof of target engagement demonstrate the great potential for use as an adjunctive treatment of lung infections in humans.

Legumain Activity Is Controlled by Extended Active Site Residues and Substrate Conformation.

Legumain is a lysosomal cysteine protease with strict specificity for cleaving after asparagine residues. By sequence comparison, legumain belongs to MEROPS clan CD of the cysteine proteases, which indicates its structural and mechanistic relation to caspases. Contrasting caspases, legumain harbors a pH-dependent ligase activity in addition to the protease activity. Although we already have a significant body of knowledge on the catalytic activities of legumain, many mechanistic details are still elusive. In this study, we provide evidence that extended active site residues and substrate conformation are steering legumain activities. Biochemical experiments and bioinformatics analysis showed that the catalytic Cys189 and His148 residues are regulated by sterically close Glu190, Ser215 and Asn42 residues. While Glu190 serves as an activity brake, Ser215 and Asn42 have a favorable effect on legumain protease activity. Mutagenesis studies using caspase-9 as model enzyme additionally showed that a similar Glu190 activity brake is also implemented in the caspases. Furthermore, we show that the substrate's conformational flexibility determines whether it will be hydrolyzed or ligated by legumain. The functional understanding of the extended active site residues and of substrate prerequisites will allow us to engineer proteases with increased enzymatic activity and better ligase substrates, with relevance for biotechnological applications.

Discovery and Characterization of Synthesized and FDA-Approved Inhibitors of Clostridial and Bacillary Collagenases.

In view of the worldwide antimicrobial resistance (AMR) threat, new bacterial targets and anti-infective agents are needed. Since important roles in bacterial pathogenesis have been demonstrated for the collagenase H and G (ColH and ColG) from Clostridium histolyticum, collagenase Q1 and A (ColQ1 and ColA) from Bacillus cereus represent attractive antivirulence targets. Furthermore, repurposing FDA-approved drugs may assist to tackle the AMR crisis and was addressed in this work. Here, we report on the discovery of two potent and chemically stable bacterial collagenase inhibitors: synthesized and FDA-approved diphosphonates and hydroxamates. Both classes showed high in vitro activity against the clostridial and bacillary collagenases. The potent diphosphonates reduced B. cereus-mediated detachment and death of cells and Galleria mellonella larvae. The hydroxamates were also tested in a similar manner; they did not have an effect in infection models. This might be due to their fast binding kinetics to bacterial collagenases.

Structural and functional studies of legumain-mycocypin complexes revealed a competitive, exosite-regulated mode of interaction.

Under pathophysiologic conditions such as Alzheimer's disease and cancer, the endolysosomal cysteine protease legumain was found to translocate to the cytosol, the nucleus, and the extracellular space. These noncanonical localizations demand for a tight regulation of legumain activity, which is in part conferred by protein inhibitors. While there is a significant body of knowledge on the interaction of human legumain with endogenous cystatins, only little is known on its regulation by fungal mycocypins. Mycocypins are characterized by (i) versatile, plastic surface loops allowing them to inhibit different classes of enzymes and (ii) a high resistance toward extremes of pH and temperature. These properties make mycocypins attractive starting points for biotechnological and medical applications. In this study, we show that mycocypins utilize an adaptable reactive center loop to target the active site of legumain in a substrate-like manner. The interaction was further stabilized by variable, isoform-specific exosites, converting the substrate recognition into inhibition. Additionally, we found that selected mycocypins were capable of covalent complex formation with legumain by forming a disulfide bond to the active site cysteine. Furthermore, our inhibition studies with other clan CD proteases suggested that mycocypins may serve as broad-spectrum inhibitors of clan CD proteases. Our studies uncovered the potential of mycocypins as a new scaffold for drug development, providing the basis for the design of specific legumain inhibitors.

The Fluorescent Enzyme Cascade Detects Low Abundance Protein Modifications Suitable for the Assembly of Functionally Annotated Modificatome Databases.

Pathophysiological functions of proteins critically depend on both their chemical composition, including post-translational modifications, and their three-dimensional structure, commonly referred to as structure-activity relationship. Current analytical methods, like capillary electrophoresis or mass spectrometry, suffer from limitations, such as the detection of unexpected modifications at low abundance and their insensitivity to conformational changes. Building on previous enzyme-based analytical methods, we here introduce a fluorescence-based enzyme cascade (fEC), which can detect diverse chemical and conformational variations in protein samples and assemble them into digital databases. Together with complementary analytical methods an automated fEC analysis established unique modification-function relationships, which can be expanded to a proteome-wide scale, i. e. a functionally annotated modificatome. The fEC offers diverse applications, including hypersensitive biomarker detection in complex samples.

Production of Functional Plant Legumain Proteases Using the Leishmania tarentolae Expression System.

Plant proteases of the legumain-type are key players in many processes along the plant life cycle. In particular, legumains are especially important in plant programmed cell death and the processing and maturation of seed storage proteins within the vacuole. Plant legumains are therefore synonymously called vacuolar processing enzymes (VPEs). Because of their dual protease and cyclase activities, plant legumains are of great interest to biotechnological applications, e.g., for the development of cyclic peptides for drug design. Despite this high interest by the scientific community, the recombinant expression of plant legumains proved challenging due to several posttranslational modifications, including (1) the formation of structurally critical disulfide bonds, (2) activation via pH-dependent proteolytic processing, and (3) stabilization by varying degrees of glycosylation. Recently we could show that LEXSY is a robust expression system for the production of plant legumains. Here we provide a general protocol for the recombinant expression of plant legumains in Leishmania cells. We further included detailed procedures for legumain purification, activation and subsequent activity assays and additionally note specific considerations with regard to isoform specific activation intermediates. This protocol serves as a universal strategy for different legumain isoforms from different source organisms.

Structure-Based Design of α-Substituted Mercaptoacetamides as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa.

Antivirulence therapy has become a widely applicable method for fighting infections caused by multidrug-resistant bacteria. Among the many virulence factors produced by the Gram-negative bacterium Pseudomonas aeruginosa, elastase (LasB) stands out as an important target as it plays a pivotal role in the invasion of the host tissue and evasion of the immune response. In this work, we explored the recently reported LasB inhibitor class of α-benzyl-N-aryl mercaptoacetamides by exploiting the crystal structure of one of the compounds. Our exploration yielded inhibitors that maintained inhibitory activity, selectivity, and increased hydrophilicity. These inhibitors were found to reduce the pathogenicity of the bacteria and to maintain the integrity of lung and skin cells in the diseased state. Furthermore, two most promising compounds increased the survival rate of Galleria mellonella larvae treated with P. aeruginosa culture supernatant.

Dichlorophenylpyridine-Based Molecules Inhibit Furin through an Induced-Fit Mechanism.

Inhibitors of the proprotein convertase furin might serve as broad-spectrum antiviral therapeutics. High cellular potency and antiviral activity against acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported for (3,5-dichlorophenyl)pyridine-derived furin inhibitors. Here we characterized the binding mechanism of this inhibitor class using structural, biophysical, and biochemical methods. We established a MALDI-TOF-MS-based furin activity assay, determined IC50 values, and solved X-ray structures of (3,5-dichlorophenyl)pyridine-derived compounds in complex with furin. The inhibitors induced a substantial conformational rearrangement of the active-site cleft by exposing a central buried tryptophan residue. These changes formed an extended hydrophobic surface patch where the 3,5-dichlorophenyl moiety of the inhibitors was inserted into a newly formed binding pocket. Consistent with these structural rearrangements, we observed slow off-rate binding kinetics and strong structural stabilization in surface plasmon resonance and differential scanning fluorimetry experiments, respectively. The discovered furin conformation offers new opportunities for structure-based drug discovery.

Inhibition of Collagenase Q1 of Bacillus cereus as a Novel Antivirulence Strategy for the Treatment of Skin-Wound Infections.

Despite the progress in surgical techniques and antibiotic prophylaxis, opportunistic wound infections with Bacillus cereus remain a public health problem. Secreted toxins are one of the main factors contributing to B. cereus pathogenicity. A promising strategy to treat such infections is to target these toxins and not the bacteria. Although the exoenzymes produced by B. cereus are thoroughly investigated, little is known about the role of B. cereus collagenases in wound infections. In this report, the collagenolytic activity of secreted collagenases (Col) is characterized in the B. cereus culture supernatant (csn) and its isolated recombinantly produced ColQ1 is characterized. The data reveals that ColQ1 causes damage on dermal collagen (COL). This results in gaps in the tissue, which might facilitate the spread of bacteria. The importance of B. cereus collagenases is also demonstrated in disease promotion using two inhibitors. Compound 2 shows high efficacy in peptidolytic, gelatinolytic, and COL degradation assays. It also preserves the fibrillar COLs in skin tissue challenged with ColQ1, as well as the viability of skin cells treated with B. cereus csn. A Galleria mellonella model highlights the significance of collagenase inhibition in vivo.

The nanotopography of SiO2 particles impacts the selectivity and 3D fold of bound allergens.

A detailed description of the changes that occur during the formation of protein corona represents a fundamental question in nanoscience, given that it not only impacts the behaviour of nanoparticles but also affects the bound proteins. Relevant questions include whether proteins selectively bind particles, whether a specific orientation is preferred for binding, and whether particle binding leads to a modulation of their 3D fold. For allergens, it is important to answer these questions given that all these effects can modify the allergenic response of atopic individuals. These potential impacts on the bound allergen are closely related to the specific properties of the involved nanoparticles. One important property influencing the formation of protein corona is the nanotopography of the particles. Herein, we studied the effect of nanoparticle porosity on allergen binding using mesoporous and non-porous SiO2 NPs. We investigated (i) the selectivity of allergen binding from a mixture such as crude pollen extract, (ii) whether allergen binding results in a preferred orientation, (iii) the influence of binding on the conformation of the allergen, and (iv) how the binding affects the allergenic response. Nanotopography was found to play a major role in the formation of protein corona, impacting the physicochemical and biological properties of the NP-bound allergen. The porosity of the surface of the SiO2 nanoparticles resulted in a higher binding capacity with pronounced selectivity for (preferentially) binding the major birch pollen allergen Bet v 1. Furthermore, the binding of Bet v 1 to the mesoporous rather than the non-porous SiO2 nanoparticles influenced the 3D fold of the protein, resulting in at least partial unfolding. Consequently, this conformational change influenced the allergenic response, as observed by mediator release assays employing the sera of patients and immune effector cells. For an in-depth understanding of the bio-nano interactions, the properties of the particles need to be considered not only regarding the identity and morphology of the material, but also their nanotopography, given that porosity may greatly influence the structure, and hence the biological behaviour of the bound proteins. Thus, thorough structural investigations upon the formation of protein corona are important when considering immunological outcomes, as particle binding can influence the allergenic response elicited by the bound allergen.

The Peptide Ligase Activity of Human Legumain Depends on Fold Stabilization and Balanced Substrate Affinities.

Protein modification by enzymatic breaking and forming of peptide bonds significantly expands the repertoire of genetically encoded protein sequences. The dual protease-ligase legumain exerts the two opposing activities within a single protein scaffold. Primarily localized to the endolysosomal system, legumain represents a key enzyme in the generation of antigenic peptides for subsequent presentation on the MHCII complex. Here we show that human legumain catalyzes the ligation and cyclization of linear peptides at near-neutral pH conditions, where legumain is intrinsically unstable. Conformational stabilization significantly enhanced legumain's ligase activity, which further benefited from engineering the prime substrate recognition sites for improved affinity. Additionally, we provide evidence that specific legumain activation states allow for differential regulation of its activities. Together these results set the basis for engineering legumain proteases and ligases with applications in biotechnology and drug development.

Structural Alterations of Antigens at the Material Interface: An Early Decision Toolbox Facilitating Safe-by-Design Nanovaccine Development.

Nanomaterials have found extensive interest in the development of novel vaccines, as adjuvants and/or carriers in vaccination platforms. Conjugation of protein antigens at the particle surface by non-covalent adsorption is the most widely used approach in licensed particulate vaccines. Hence, it is essential to understand proteins' structural integrity at the material interface in order to develop safe-by-design nanovaccines. In this study, we utilized two model proteins, the wild-type allergen Bet v 1 and its hypoallergenic fold variant (BM4), to compare SiO2 nanoparticles with Alhydrogel® as particulate systems. A set of biophysical and functional assays including circular dichroism spectroscopy and proteolytic degradation was used to examine the antigens' structural integrity at the material interface. Conjugation of both biomolecules to the particulate systems decreased their proteolytic stability. However, we observed qualitative and quantitative differences in antigen processing concomitant with differences in their fold stability. These changes further led to an alteration in IgE epitope recognition. Here, we propose a toolbox of biophysical and functional in vitro assays for the suitability assessment of nanomaterials in the early stages of vaccine development. These tools will aid in safe-by-design innovations and allow fine-tuning the properties of nanoparticle candidates to shape a specific immune response.

OFF-State-Specific Inhibition of the Proprotein Convertase Furin.

The pro-protein convertase furin is a highly specific serine protease involved in the proteolytic maturation of many proteins in the secretory pathway. It also activates surface proteins of many viruses including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furin inhibitors effectively suppress viral replication and thus are promising antiviral therapeutics with broad application potential. Polybasic substrate-like ligands typically trigger conformational changes shifting furin's active site cleft from the OFF-state to the ON-state. Here, we solved the X-ray structures of furin in complex with four different arginine mimetic compounds with reduced basicity. These guanylhydrazone-based inhibitor complexes showed for the first time an active site-directed binding mode to furin's OFF-state conformation. The compounds undergo unique interactions within the S1 pocket, largely different compared to substrate-like ligands. A second binding site was identified at the S4/S5 pocket of furin. Crystallography-based titration experiments confirmed the S1 site as the primary binding pocket. We also tested the proprotein convertases PC5/6 and PC7 for inhibition by guanylhydrazones and found an up to 7-fold lower potency for PC7. Interestingly, the observed differences in the Ki values correlated with the sequence conservation of the PCs at the allosteric sodium binding site. Therefore, OFF-state-specific targeting of furin can serve as a valuable strategy for structure-based development of PC-selective small-molecule inhibitors.

Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium.

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa-soluble tissue factor (TF) complex was solved at 1.8 Šresolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163-180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.