Comparison of natural and recombinant clitocypins, the fungal cysteine protease inhibitors

A member of the cysteine protease inhibitor clitocypin gene family from basidiomycete Clitocybe nebularis was expressed in Escherichia coli. Following careful optimization of the expression procedure the active inhibitor was purified from inclusion bodies and its properties examined and compared to those of the natural clitocypin. The CD spectrum of recombinant clitocypin was similar to that of natural clitocypin, indicating that protein was properly refolded during purification. In spite of some differences in primary structure, structural, functional and immunological equivalence was established. Kinetic analyses of the natural and recombinant clitocypins were performed. Both clitocypins inhibited a range of cysteine proteases to a similar extent, and demonstrated an unusually broad inhibitory spectrum, including distantly related proteases, such as papain and legumain, belonging to different protease families. The homogenous, biologically active recombinant clitocypin is obtained at levels adequate for further structure-function studies.     

Human rhinovirus 3C protease: a cysteine protease showing the trypsin(ogen)-like fold

Viral-encoded proteases cleave precursor polyprotein(s) leading to maturation of infectious virions. Strikingly, human rhinovirus 3C protease shows the trypsin(ogen)-like serine protease fold based on two topologically equivalent six-stranded β-barrels, but displays residue Cys147 as the active site nucleophile. By contrast, papain, which is representative of most cysteine proteases, does not display the trypsin(ogen)-like fold. Remarkably, in human rhinovirus 3C cysteine protease, the catalytic residues Cys147, His40 and Glu71 are positioned as Ser195, His57 and Asp102, respectively, building up the catalytic triad of serine proteases in the chymotrypsin–trypsin–elastase family. However, as compared to trypsin-like serine proteases and their zymogens, residue His40 and the oxyanion hole of human rhinovirus 3C cysteine protease, both key structural components of the active site, are located closer to the protein core. Human rhinovirus 3C cysteine protease cleaves preferentially GlnGly peptide bonds or, less commonly, the GlnSer, GlnAla, GluSer or GluGly pairs. Finally, human rhinovirus 3C cysteine protease and the 3CD cysteine protease–polymerase covalent complex bind the 5′ non-coding region of rhinovirus genomic RNA, an essential function for replication of the viral genome.     

Mastering the canonical loop of serine protease inhibitors: enhancing potency by optimising the internal hydrogen bond network

Background

Canonical serine protease inhibitors commonly bind to their targets through a rigid loop stabilised by an internal hydrogen bond network and disulfide bond(s). The smallest of these is sunflower trypsin inhibitor (SFTI-1), a potent and broad-range protease inhibitor. Recently, we re-engineered the contact β-sheet of SFTI-1 to produce a selective inhibitor of kallikrein-related peptidase 4 (KLK4), a protease associated with prostate cancer progression. However, modifications in the binding loop to achieve specificity may compromise structural rigidity and prevent re-engineered inhibitors from reaching optimal binding affinity.

Methodology/Principal Findings

In this study, the effect of amino acid substitutions on the internal hydrogen bonding network of SFTI were investigated using an in silico screen of inhibitor variants in complex with KLK4 or trypsin. Substitutions favouring internal hydrogen bond formation directly correlated with increased potency of inhibition in vitro. This produced a second generation inhibitor (SFTI-FCQR Asn₁₄) which displayed both a 125-fold increased capacity to inhibit KLK4 (K _i = 0.0386±0.0060 nM) and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn₁₄ was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion.

Conclusion/Significance

These findings highlight the importance of conserving structural rigidity of the binding loop in addition to optimising protease/inhibitor contacts when re-engineering canonical serine protease inhibitors.     

Basidiomycetes harbour a hidden treasure of proteolytic diversity

This survey is the first to investigate the proteolytic potential of a large number of basidiomycetes. Aqueous extracts of 43 basidiomycetes were investigated for their content of proteolytic activities, using gelatin zymography. The activities were characterised qualitatively using class specific inhibitors. All four catalytic classes of proteases were present, with 4% of all activities classified as aspartic, 5% as cysteine, 6% as metallo and 22% as serine proteases, while the remaining activities could not be assigned unambiguously. The majority of the latter were not inhibited by any of the inhibitors used and were termed insensitive. Different proteolytic activities are evenly distributed among members of all orders of basidiomycetes, although some taxa are a richer source of proteases than others. A significant number of the cysteine protease activities shown here have not previously been reported in basidiomycetes. The fungal cysteine and serine protease inhibitors, clitocypin and CNSPI (Clitocybe nebularis serine protease inhibitor), both inhibited a number of activities and even a few activities that were otherwise insensitive to all other inhibitors used, hence indicating their potential for a regulatory role. The number and diversity of proteases in basidiomycetes are seen to be remarkable and encourage further investigation.     

Novel non-peptidic vinylsulfones targeting the S2 and S3 subsites of parasite cysteine proteases

We describe here the identification of non-peptidic vinylsulfones that inhibit parasite cysteine proteases in vitro and inhibit the growth of Trypanosoma brucei brucei parasites in culture. A high resolution (1.75 Å) co-crystal structure of 8a bound to cruzain reveals how the non-peptidic P2/P3 moiety in such analogs bind the S2 and S3 subsites of the protease, effectively recapitulating important binding interactions present in more traditional peptide-based protease inhibitors and natural substrates.     

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 Å resolution were obtained using hanging drop method by vapor diffusion at 18 °C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function.     

Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus

Staphylococcus aureus is responsible for a variety of human infections, including life-threatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in particular SplA protease, remain poorly characterized. Here, we report development of specific inhibitors of SplA protease. The design, synthesis, and activity of a series of α-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding modes of the high-affinity compounds Cbz-Phe^P-(OC₆H₄−4-SO₂CH₃)₂ and Suc-Val-Pro-Phe^P-(OC₆H₅)₂ are revealed by high-resolution crystal structures of complexes with the protease. Surprisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of α-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases.     

Modes of inhibition of cysteine proteases

Cysteine proteases are involved in many physiological processes and their hyperactivity may lead to severe diseases. Nature has developed various strategies to protect cells and whole organisms against undesired proteolysis. One of them is the control of proteolytic activity by inhibition. This paper presents the mechanisms underlying the action of proteinaceous inhibitors of cysteine proteinases and covers propeptides binding backwards relative to the substrate or distorting the protease catalytic centre similarly to serpins, the p35 protein binding covalently to the enzyme, and cystatins that are exosite binding inhibitors. The paper also discusses tyropins and chagasins that, although unrelated to cystatins, inhibit cysteine proteinases by a similar mechanism, as well as inhibitors of the apoptosis protein family that bind in a direction opposite to that of the substrate, similarly to profragments. Special attention is given to staphostatins, a novel family of inhibitors acting in an unusual manner.     

A programmed cell death pathway in the malaria parasite Plasmodium falciparum has general features of mammalian apoptosis but is mediated by clan CA cysteine proteases

Several recent discoveries of the hallmark features of programmed cell death (PCD) in Plasmodium falciparum have presented the possibility of revealing novel targets for antimalarial therapy. Using a combination of cell-based assays, flow cytometry and fluorescence microscopy, we detected features including mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in parasites induced with chloroquine (CQ) and staurosporine (ST). The use of the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (zVAD), and the mitochondria outer membrane permeabilization (MOMP) inhibitor, 4-hydroxy-tamoxifen, enabled the characterization of a novel CQ-induced pathway linking cysteine protease activation to downstream mitochondrial dysregulation, amplified protease activity and DNA fragmentation. The PCD features were observed only at high (μM) concentrations of CQ. The use of a new synthetic coumarin-labeled chloroquine (CM-CQ) showed that these features may be associated with concentration-dependent differences in drug localization. By further using cysteine protease inhibitors z-Asp-Glu-Val-Asp-fmk (zDEVD), z-Phe-Ala-fmk (zFA), z-Phe-Phe-fmk (zFF), z-Leu-Leu-Leu-fmk (zLLL), E64d and CA-074, we were able to implicate clan CA cysteine proteases in CQ-mediated PCD. Finally, CQ induction of two CQ-resistant parasite strains, 7G8 and K1, reveals the existence of PCD features in these parasites, the extent of which was less than 3D7. The use of the chemoreversal agent verapamil implicates the parasite digestive vacuole in mediating CQ-induced PCD.     

Structure of the autocatalytic cysteine protease domain of potyvirus helper-component proteinase

The helper-component proteinase (HC-Pro) of potyvirus is involved in polyprotein processing, aphid transmission, and suppression of antiviral RNA silencing. There is no high resolution structure reported for any part of HC-Pro, hindering mechanistic understanding of its multiple functions. We have determined the crystal structure of the cysteine protease domain of HC-Pro from turnip mosaic virus at 2.0 Å resolution. As a protease, HC-Pro only cleaves a Gly-Gly dipeptide at its own C terminus. The structure represents a postcleavage state in which the cleaved C terminus remains tightly bound at the active site cleft to prevent trans activity. The structure adopts a compact α/β-fold, which differs from papain-like cysteine proteases and shows weak similarity to nsP2 protease from Venezuelan equine encephalitis alphavirus. Nevertheless, the catalytic cysteine and histidine residues constitute an active site that is highly similar to these in papain-like and nsP2 proteases. HC-Pro recognizes a consensus sequence YXVGG around the cleavage site between the two glycine residues. The structure delineates the sequence specificity at sites P1–P4. Structural modeling and covariation analysis across the Potyviridae family suggest a tryptophan residue accounting for the glycine specificity at site P1′. Moreover, a surface of the protease domain is conserved in potyvirus but not in other genera of the Potyviridae family, likely due to extra functional constrain. The structure provides insight into the catalysis mechanism, cis-acting mode, cleavage site specificity, and other functions of the HC-Pro protease domain.     

SjAPI,the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

Background

Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear.

Principal Findings

Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI), Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2), Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI), and Buthus martensii Ascaris-type protease inhibitor (BmAPI). The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues “AAV” and might be a useful template to produce new serine protease inhibitors.

Conclusions/Significance

To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.     

The structure of chagasin in complex with a cysteine protease clarifies the binding mode and evolution of an inhibitor family

Protein inhibitors of proteolytic enzymes regulate proteolysis and prevent the pathological effects of excess endogenous or exogenous proteases. Cysteine proteases are a large family of enzymes found throughout the plant and animal kingdoms. Disturbance of the equilibrium between cysteine proteases and natural inhibitors is a key event in the pathogenesis of cancer, rheumatoid arthritis, osteoporosis, and emphysema. A family (I42) of cysteine protease inhibitors (http://merops.sanger.ac.uk) was discovered in protozoan parasites and recently found widely distributed in prokaryotes and eukaryotes. We report the 2.2 A crystal structure of the signature member of the I42 family, chagasin, in complex with a cysteine protease. Chagasin has a unique variant of the immunoglobulin fold with homology to human CD8alpha. Interactions of chagasin with a target protease are reminiscent of the cystatin family inhibitors. Protein inhibitors of cysteine proteases may have evolved more than once on nonhomologous scaffolds.     

Heterogeneity in the cysteine protease inhibitor clitocypin gene family

Clitocypin from the basidiomycete Clitocybe nebularis is the first fungal protein cysteine protease inhibitor to be characterised in detail, yet no information on its molecular genetics is available. Owing to its unique characteristics, it was assigned as the only member of a new family of cysteine protease inhibitors in the MEROPS inhibitor classification. Here we describe the full-length sequence of the clitocypin gene. A BLAST search confirmed its lack of significant sequence similarity to any other gene. The gene is composed of four exons and three short introns and belongs to a small family of closely related genes with more than 90% identity. Sequence variability is evenly distributed in introns and exons and deduced amino acid substitutions are distributed throughout the protein sequence. Basidiocarps collected at two distant locations were examined and the level of heterogeneity found in one basidiocarp is similar to that between the two. Sequencing of the ribosomal DNA spacers from the two basidiocarps confirmed that the heterogeneity observed in the clitocypin gene is not due to evolutionary divergence of the two specimens caused by geographic separation. Clitocypin is expressed in different parts of the basidiocarp and in cultured mycelia in a manner suggesting regulation by developmental and/or environmental factors.     

Partial purification and characterization of a cysteine protease inhibitor from the plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.     

Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases

Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys^2nd and Cys^6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be applied in both health care and agricultural industries, and could lead to new methods for breeding fungus-resistant transgenic crops and antifungal transgenic silkworm strains.     

Crystal Structure of Der f 7, a Dust Mite Allergen from Dermatophagoides farinae

Background

Der f 7 is the group 7 allergen from the dust mite Dermatophagoides farinae, homologous to the major allergen Der p 7 from D. pteronyssinus. Monoclonal antibody that bind to residues Leu48 and Phe50 was found to inhibit IgE binding to residue Asp159, which is important for the cross-reactivity between Der f 7 and Der p 7.

Methodology/Principal Findings

Here, we report the crystal structure of Der f 7 that shows an elongated and curved molecule consisting of two anti-parallel β-sheets – one 4-stranded and the other 5-stranded – that wrap around a long C-terminal helix. The overall fold of Der f 7 is similar to Der p 7 but key difference was found in the β1–β2 loop region. In Der f 7, Leu48 and Phe50 are in close proximity to Asp159, explaining why monoclonal antibody binding to Leu48 and Phe50 can inhibit IgE binding to Asp159. Both Der f 7 and Der p 7 bind weakly to polymyxin B via a similar binding site that is formed by the N-terminal helix, the 4-stranded β-sheet and the C-terminal helix. The thermal stability of Der f 7 is significantly lower than that of Der p 7, and the stabilities of both allergens are highly depend on pH.

Conclusion/Significance

Der f 7 is homologous to Der p 7 in terms of the amino acid sequence and overall 3D structure but with significant differences in the region proximal to the IgE epitope and in thermal stability. The crystal structure of Der f 7 provides a basis for studying the function and allergenicity of this group of allergens.     

Cross-Talk between Malarial Cysteine Proteases and Falstatin: The BC Loop as a Hot-Spot Target

Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.     

The plasticity of the β-trefoil fold constitutes an evolutionary platform for protease inhibition

Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition.     

Crystal structure of human cathepsin V

Cathepsin V is a lysosomal cysteine protease that is expressed in the thymus, testis and corneal epithelium. We have determined the 1.6 A resolution crystal structure of human cathepsin V associated with an irreversible vinyl sulfone inhibitor. The fold of this enzyme is similar to the fold adopted by other members of the papain superfamily of cysteine proteases. This study provides a framework for understanding the structural basis for cathepsin V's activity and will aid in the design of inhibitors of this enzyme. A comparison of cathepsin V's active site with the active sites of related proteases revealed a number of differences, especially in the S2 and S3 subsites, that could be exploited in identifying specific cathepsin V inhibitors or in identifying inhibitors of other cysteine proteases that would be selective against cathepsin V.     

Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: functional convergence of a common protein fold

Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight β-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.