The enzymatic activation of human legumain requires both proteolytic cleavage and conformational reordering and is modulated by its substrate as well as cofactors. These biochemical findings are aided by the crystallization and initial crystallographic analysis of legumain.
Localized mainly to endo/lysosomes, legumain plays an important role in exogenous antigen processing and presentation. The cysteine protease legumain, also known as asparaginyl endopepetidase AEP, is synthesized as a zymogen and is known to undergo pH-dependent autoproteolytic activation whereby N-terminal and C-terminal propeptides are released. However, important mechanistic details of this pH-dependent activation as well as the characteristic pH activity profile remain unclear. Here, it is shown that all but one of the autocatalytic cleavage events occur in trans, with only the release of the C-terminal propeptide being relevant to enzymatic activity. An intriguing super-activation event that appears to be exclusively conformational in nature and enhances the enzymatic activity of proteolytically fully processed legumain by about twofold was also found. Accepting asparagines and, to lesser extent, aspartic acid in P1, super-activated legumain exhibits a marked pH dependence that is governed by the P1 residue of its substrate and conformationally stabilizing factors such as temperature or ligands. The crystallization and preliminary diffraction data analysis of active legumain are presented, which form an important basis for further studies that should clarify fundamental aspects of activation, activity and inactivation of legumain, which is a key target in (auto-)immunity and cancer.
cysteine proteases; pH regulation; conformational activation; substrate-dependent activity; autoimmune diseases; cancer
•Histidine-tagged bovine enterokinase was refolded from bacterial inclusion bodies.•Refolding yields satisfy high demands of protein crystallography projects.•Enterokinase specifically cleaved artificial propeptides from target proteins.
Enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its N-terminal propeptide. Due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate N-terminal affinity tags from target proteins with authentic N-termini. In order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase light chain. The purified, highly active enzyme successfully processed recombinant target proteins, while the pentahistidine-tag facilitated post-cleavage removal. Hence, we conclude that producing enterokinase in one's own laboratory is an efficient alternative to the commercial enzyme.
BENAC, benzamidine affinity chromatography; EK, enterokinase; GST, glutathione-S-transferase; IB, inclusion body; IEC, ion exchange chromatography; IMAC, mmobilized metal ion affinity chromatography; KLK, kallikrein-related peptidase; SUMO, small ubiquitin-like modifier; TEV, tobacco etch virus; uPA, urokinase-type plasminogen activator; Biotechnology; Enteropeptidase; Inclusion bodies; In vitro folding; Serine proteases
Background: Frequently reported dimerization of allergens may contribute to their allergenicity.
Results: Polysulfide-bridged allergen dimers exhibit different allergenic properties compared with the monomer.
Conclusion: The N-terminal region has a distinct susceptibility for modifications and impacts its protein-protein interaction characteristics.
Significance: The crystal structures well mimic transient dimerization of the allergens in solution, providing a rational for effective IgE cross-linking on effector cells.
Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.
Allergen; Crystal Structure; Mass Spectrometry (MS); Post-translational Modification; Protein Assembly; Dimerization; Noncanonical Amino Acid Incorporation; Polysulfide Linking; Position-specific Alteration of Genetic Code
Npro is a key effector protein of pestiviruses such as bovine viral diarrhea virus and abolishes host cell antiviral defense mechanisms. Synthesized as the N-terminal part of the viral polyprotein, Npro releases itself via an autoproteolytic cleavage, triggering its immunological functions. However, the mechanisms of its proteolytic action and its immune escape were unclear. Here, we present the crystal structures of Npro to 1.25 Å resolution. Structures of pre- and postcleavage intermediates identify three catalytically relevant elements. The trapping of the putative catalytic water reveals its distinct roles as a base, acid, and nucleophile. The presentation of the substrate further explains the enigmatic latency of the protease, ensuring a single in cis cleavage. Additionally, we identified a zinc-free, disulfide-linked conformation of the TRASH motif, an interaction hub of immune factors. The structure opens additional opportunities in utilizing Npro as an autocleaving fusion protein and as a pharmaceutical target.
•Putative catalytic water reveals distinct roles as a base, acid, and nucleophile•The structural mechanism explains a single in cis cleavage•The bimodular architecture reflects proteolytic and immunological functions•The structure provides two orthogonal targets for therapy
The pestivirus protease Npro abolishes host cell antiviral defense. Structures by Zögg et al. reveal residues that act as nucleophiles and as oxyanion pockets. The trapped catalytic water has distinct roles as base, acid, and nucleophile, and the substrate binding mode explains the single in cis cleavage.
Background: Bacterial collagenases degrade collagen substrates with high efficiency yet varying specificity.
Results: The newly identified calcium site, aspartate switch, and conformational selectivity filter regulate substrate access to the active sites of these collagenases.
Conclusion: The unanticipated dynamics of the substrate recognition sites plus zinc occupancy combine to tune the enzymatic activity.
Significance: The crystal structures provide a rational framework to understand and optimize the isoform-dependent collagenase activities.
Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein in the biosphere. Here we present crystal structures of the peptidases of three clostridial collagenase isoforms (ColG, ColH, and ColT). The comparison of unliganded and liganded structures reveals a quaternary subdomain dynamics. In the unliganded ColH structure, this globular dynamics is modulated by an aspartate switch motion that binds to the catalytic zinc. We further identified a calcium binding site in proximity to the catalytic zinc. Both ions are required for full activity, explaining why calcium critically affects the enzymatic activity of clostridial collagenases. Our studies further reveal that loops close to the active site thus serve as characteristic substrate selectivity filter. These elements explain the distinct peptidolytic and collagenolytic activities of these enzymes and provide a rational framework to engineer collagenases with customized substrate specificity as well as for inhibitor design.
Protease; Protein Degradation; Protein Structure; Proteolytic Enzymes; X-ray Crystallography; Collagenase; Metal Regulation
The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.
Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.
The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.
► Ligand binding to Bet v 1 may contribute to explain its allergenicity. ► High-resolution structures reveal the binding mode of diverse ligands to Bet v 1. ► Residue 30 starkly influences the binding properties of different Bet v 1 isoforms. ► Ternary complexes with diverse ligands explain anomalous fluorescence binding curves. ► Betv1 isoforms differ in ligand binding, which may translate into their allergenicity.
ANS, 1-anilino-8-naphthalene sulfonate; BRA, brassinolide; DXC, deoxycholate; iDXC, inner deoxycholate; oDXC, outer deoxycholate; LPS, lipopolysaccharide; MPD, 2-methyl-2,4-pentanediol; NDSB-256, non-detergent sulfobetaine 256; PR-10, pathogenesis-related protein 10; PDB, Protein Data Bank; molecular allergenicity; ANS displacement assay; structure–allergenicity relationship; binding specificity and promiscuity; dressed allergens
Collagen constitutes one third of the body protein in humans, reflecting its extraordinary role in health and disease. Of similar importance, therefore, are the idiosyncratic proteases that nature evolved for collagen remodeling. Intriguingly, the most efficient collagenases are those that enable clostridial bacteria to colonize their host tissues, but despite intense studies, the structural and mechanistic basis of these enzymes has remained elusive. Here we present the crystal structure of collagenase G from Clostridium histolyticum at 2.55 Å resolution. By combining the structural data with enzymatic and mutagenesis studies, we derive a conformational two-state model of bacterial collagenolysis, in which the recognition and unraveling of collagen microfibrils into triple helices as well as the unwinding of the latter go hand in hand with collagenase opening and closing.
Recurring groups of atoms in molecules are surrounded by specific canonical distributions of electrons. Deviations from these distributions reveal unrealistic molecular geometries. Here, we show how canonical electron densities can be combined with classical electron densities derived from X-ray diffraction experiments to drive the real space refinement of crystal structures. The refinement process generally yields superior molecular models with reduced excess electron densities and improved stereochemistry without compromising the agreement between molecular models and experimental data.
► Recurring groups of atoms in proteins are surrounded by canonical electron densities ► Deviations from canonical densities reveal unrealistic molecular geometries ► Canonical density refinement removes electron excess and improves stereochemistry
Including the true tissue kallikrein KLK1, kallikrein-related peptidases (KLKs) represent a family of fifteen mammalian serine proteases. While the physiological roles of several KLKs have been at least partially elucidated, their activation and regulation remain largely unclear. This obscurity may be related to the fact that a given KLK fulfills many different tasks in diverse fetal and adult tissues, and consequently, the timescale of some of their physiological actions varies significantly. To date, a variety of endogenous inhibitors that target distinct KLKs have been identified. Among them are the attenuating Zn2+ ions, active site-directed proteinaceous inhibitors, such as serpins and the Kazal-type inhibitors, or the huge, unspecific compartment forming α2-macroglobulin. Failure of these inhibitory systems can lead to certain pathophysiological conditions. One of the most prominent examples is the Netherton syndrome, which is caused by dysfunctional domains of the Kazal-type inhibitor LEKTI-1 which fail to appropriately regulate KLKs in the skin. Small synthetic inhibitory compounds and natural polypeptidic exogenous inhibitors have been widely employed to characterize the activity and substrate specificity of KLKs and to further investigate their structures and biophysical properties. Overall, this knowledge leads not only to a better understanding of the physiological tasks of KLKs, but is also a strong fundament for the synthesis of small compound drugs and engineered biomolecules for pharmaceutical approaches. In several types of cancer, KLKs have been found to be overexpressed, which makes them clinically relevant biomarkers for prognosis and monitoring. Thus, down regulation of excessive KLK activity in cancer and in skin diseases by small inhibitor compounds may represent attractive therapeutical approaches.
Tissue kallikrein; Specificity pockets; Inhibitory compound; Zinc; Rule of five
The catalytic domain of collagenase G from C. histolyticum was expressed in E. coli BL21 (DE3) and purified using affinity and size-exclusion column-chromatographic methods. Crystals were obtained at 290 K by the sitting-drop vapour-diffusion method and diffraction data have been collected to 2.75 Å resolution.
The catalytic domain of collagenase G from Clostridium histolyticum has been cloned, recombinantly expressed in Escherichia coli and purified using affinity and size-exclusion column-chromatographic methods. Crystals of the catalytic domain were obtained from 0.12 M sodium citrate and 23%(v/v) PEG 3350 at 293 K. The crystals diffracted to 2.75 Å resolution using synchrotron radiation. The crystals belong to an orthorhombic space group, with unit-cell parameters a = 57, b = 109, c = 181 Å. This unit cell is consistent with the presence of one molecule per asymmetric unit and a solvent content of approximately 53%.
collagenase G; Clostridium histolyticum
Clostridial collagenases are foe and friend: on the one hand, these enzymes enable host infiltration and colonization by pathogenic clostridia, and on the other hand, they are valuable biotechnological tools due to their capacity to degrade various types of collagen and gelatine. However, the demand for high-grade preparations exceeds supply due to their pathogenic origin and the intricate purification of homogeneous isoforms. We present the establishment of an Escherichia coli expression system for a variety of constructs of collagenase G (ColG) and H (ColH) from Clostridium histolyticum and collagenase T (ColT) from Clostridium tetani, mimicking the isoforms in vivo. Based on a setup of five different expression strains and two expression vectors, 12 different constructs were expressed, and a flexible purification platform was established, consisting of various orthogonal chromatography steps adaptable to the individual needs of the respective variant. This fast, cost-effective, and easy-to-establish platform enabled us to obtain at least 10 mg of highly pure mono-isoformic protein per liter of culture, ideally suited for numerous sophisticated downstream applications. This production and purification platform paves the way for systematic screenings of recombinant collagenases to enlighten the biochemical function and to identify key residues and motifs in collagenolysis.
Clostridial collagenases; Expression; Purification; Platform
of the major birch pollen allergen Bet v 1 alters the
immune responses toward this protein, but the underlying chemical
mechanisms are not yet understood. Here we address the efficiency
and site-selectivity of the nitration reaction of recombinant protein
samples of Bet v 1.0101 with different nitrating agents relevant for
laboratory investigations (tetranitromethane, TNM), for physiological
processes (peroxynitrite, ONOO–), and for the health
effects of environmental pollutants (nitrogen dioxide and ozone, O3/NO2). We determined the total tyrosine nitration
degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array
detection and HPLC coupled to high-resolution mass spectrometry analysis
of intact proteins, HPLC coupled to tandem mass spectrometry analysis
of tryptic peptides, and amino acid analysis of hydrolyzed samples
were performed. The preferred reaction sites were tyrosine residues
at the following positions in the polypeptide chain: Y83 and Y81 for
TNM, Y150 for ONOO–, and Y83 and Y158 for O3/NO2. The tyrosine residues Y83 and Y81 are located
in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible
and flexible structures of the C-terminal region. The heterogeneous
reaction with O3/NO2 was found to be strongly
dependent on the phase state of the protein. Nitration rates were
about one order of magnitude higher for aqueous protein solutions
(∼20% per day) than for protein filter samples (∼2%
per day). Overall, our findings show that the kinetics and site-selectivity
of nitration strongly depend on the nitrating agent and reaction conditions,
which may also affect the biological function and adverse health effects
of the nitrated protein.
Bet v 1.0101; HPLC−MS/MS; tyrosine nitration; nitration sites; air pollution