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1.  Protease recognition sites in Bet v 1a are cryptic, explaining its slow processing relevant to its allergenicity 
Scientific Reports  2015;5:12707.
Despite a high similarity with homologous protein families, only few proteins trigger an allergic immune response with characteristic TH2 polarization. This puzzling observation is illustrated by the major birch pollen allergen Bet v 1a and its hypoallergenic protein isoforms, e.g., Bet v 1d. Given the key role of proteolytic processing in antigen presentation and T cell polarization, we investigated the recognition of Bet v 1 isoforms by the relevant protease cathepsin S. We found that at moderately acidic pH values Bet v 1a bound to cathepsin S with significantly lower affinity and was more slowly cleaved than its hypoallergenic isoform Bet v 1d. Only at pH values ≤4.5 the known proteolytic cleavage sites in Bet v 1a became accessible, resulting in a strong increase in affinity towards cathepsin S. Antigen processing and class II MHC loading occurs at moderately acidic compartments where processing of Bet v 1a and Bet v 1d differs distinctly. This difference translates into low and high density class II MHC loading and subsequently in TH2 and TH1 polarization, respectively.
PMCID: PMC4522599  PMID: 26235974
2.  Structure and Mechanism of an Aspartimide-Dependent Peptide Ligase in Human Legumain** 
Peptide ligases expand the repertoire of genetically encoded protein architectures by synthesizing new peptide bonds, energetically driven by ATP or NTPs. Here, we report the discovery of a genuine ligase activity in human legumain (AEP) which has important roles in immunity and tumor progression that were believed to be due to its established cysteine protease activity. Defying dogma, the ligase reaction is independent of the catalytic cysteine but exploits an endogenous energy reservoir that results from the conversion of a conserved aspartate to a metastable aspartimide. Legumain’s dual protease–ligase activities are pH- and thus localization controlled, dominating at acidic and neutral pH, respectively. Their relevance includes reversible on–off switching of cystatin inhibitors and enzyme (in)activation, and may affect the generation of three-dimensional MHC epitopes. The aspartate–aspartimide (succinimide) pair represents a new paradigm of coupling endergonic reactions in ATP-scarce environments.
PMCID: PMC4506564  PMID: 25630877
endergonic reactions; enzyme catalysis; hydrolysis; ligases; protein modification
3.  Structure-Function Analyses of Human Kallikrein-related Peptidase 2 Establish the 99-Loop as Master Regulator of Activity* 
The Journal of Biological Chemistry  2014;289(49):34267-34283.
Background: Serine proteases KLK2 and KLK3 clear the way for spermatozoa before impregnation.
Results: Enzymatic assays and structures of KLK2 elucidate its catalytic action, especially when compared with conformations of similar proteases.
Conclusion: Flexible loops around the active site of serine proteases open concertedly upon substrate binding.
Significance: This mechanistic model will stimulate the design of pharmaceutical inhibitors.
Human kallikrein-related peptidase 2 (KLK2) is a tryptic serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, a major component of seminal fluid. Most likely it activates and complements chymotryptic KLK3 (prostate-specific antigen) in cleaving seminal clotting proteins, resulting in sperm liquefaction. KLK2 belongs to the “classical” KLKs 1–3, which share an extended 99- or kallikrein loop near their non-primed substrate binding site. Here, we report the 1.9 Å crystal structures of two KLK2-small molecule inhibitor complexes. In both structures discontinuous electron density for the 99-loop indicates that this loop is largely disordered. We provide evidence that the 99-loop is responsible for two biochemical peculiarities of KLK2, i.e. reversible inhibition by micromolar Zn2+ concentrations and permanent inactivation by autocatalytic cleavage. Indeed, several 99-loop mutants of KLK2 displayed an altered susceptibility to Zn2+, which located the Zn2+ binding site at the 99-loop/active site interface. In addition, we identified an autolysis site between residues 95e and 95f in the 99-loop, whose elimination prevented the mature enzyme from limited autolysis and irreversible inactivation. An exhaustive comparison of KLK2 with related structures revealed that in the KLK family the 99-, 148-, and 220-loop exist in open and closed conformations, allowing or preventing substrate access, which extends the concept of conformational selection in trypsin-related proteases. Taken together, our novel biochemical and structural data on KLK2 identify its 99-loop as a key player in activity regulation.
PMCID: PMC4256358  PMID: 25326387
Crystallography; Enzyme Kinetics; Kallikrein; Prostate Cancer; Serine Protease; Substrate Specificity; 99-Loop; Autolytic Cleavage; Conformational Selection; Zinc Inhibition
4.  The Impact of Nitration on the Structure and Immunogenicity of the Major Birch Pollen Allergen Bet v 1.0101 
PLoS ONE  2014;9(8):e104520.
Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of TH1-priming cytokines, thus favoring a TH2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.
PMCID: PMC4134196  PMID: 25126882
5.  Structural Integrity of the Antigen Is a Determinant for the Induction of T-Helper Type-1 Immunity in Mice by Gene Gun Vaccines against E.coli Beta-Galactosidase 
PLoS ONE  2014;9(7):e102280.
The type of immune response is critical for successful protection and typically determined by pathogen-associated danger molecules. In contrast, protein antigens are usually regarded as passive target structures. Here, we provide evidence that the structure of the antigen can profoundly influence the type of response that is elicited under else identical conditions. In mice, gene gun vaccines induce predominantly Th2-biased immune reactions against most antigens. One exception is E. coli beta-galactosidase (βGal) that induces a balanced Th1/Th2 response. Because both, the delivered material (plasmid DNA-coated gold particles) as well as the procedure (biolistic delivery to the skin surface) is the same as for other antigens we hypothesized that Th1 induction could be a function of βGal protein expressed in transfected cells. To test this we examined gene gun vaccines encoding structural or functional variants of the antigen. Employing a series of gene gun vaccines encoding individual structural domains of βGal, we found that neither of them induced IgG2a antibodies. Even disruption of the homo-tetramer association of the native protein by deletion of a few N-terminal amino acids was sufficient to abrogate IgG2a production. However, enzymatically inactive βGal with only one point mutation in the catalytic center retained the ability to induce Th1 reactions. Thus, structural but not functional integrity of the antigen must be retained for Th1 induction. βGal is not a Th1 adjuvant in the classical sense because neither were βGal-transgenic ROSA26 mice particularly Th1-biased nor did co-administration of a βGal-encoding plasmid induce IgG2a against other antigens. Despite this, gene gun vaccines elicited Th1 reactions to antigens fused to the open reading frame of βGal. We interpret these findings as evidence that different skin-borne antigens may be differentially handled by the immune system and that the three-dimensional structure of an antigen is an important determinant for this.
PMCID: PMC4099185  PMID: 25025197
6.  Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions 
Pyrin domains (PYDs) recruit downstream effector molecules in NLR signalling. A specific charge-relay system suggests a the formation of a signalling complex involving a PYD dimer.
The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed.
PMCID: PMC4089490  PMID: 25004977
pyrin domain; NLRP14
7.  X-ray Structures of Human Furin in Complex with Competitive Inhibitors 
ACS Chemical Biology  2014;9(5):1113-1118.
Furin inhibitors are promising therapeutics for the treatment of cancer and numerous infections caused by bacteria and viruses, including the highly lethal Bacillus anthracis or the pandemic influenza virus. Development and improvement of inhibitors for pharmacological use require a detailed knowledge of the protease’s substrate and inhibitor binding properties. Here we present a novel preparation of human furin and the first crystal structures of this enzyme in complex with noncovalent inhibitors. We show the inhibitor exchange by soaking, allowing the investigation of additional inhibitors and substrate analogues. Thus, our work provides a basis for the rational design of furin inhibitors.
PMCID: PMC4026159  PMID: 24666235
8.  Proteomic protease specificity profiling of clostridial collagenases reveals their intrinsic nature as dedicated degraders of collagen☆☆☆ 
Journal of Proteomics  2014;100(100):102-114.
Clostridial collagenases are among the most efficient degraders of collagen. Most clostridia are saprophytes and secrete proteases to utilize proteins in their environment as carbon sources; during anaerobic infections, collagenases play a crucial role in host colonization. Several medical and biotechnological applications have emerged utilizing their high collagenolytic efficiency. However, the contribution of the functionally most important peptidase domain to substrate specificity remains unresolved. We investigated the active site sequence specificity of the peptidase domains of collagenase G and H from Clostridium histolyticum and collagenase T from Clostridium tetani. Both prime and non-prime cleavage site specificity were simultaneously profiled using Proteomic Identification of protease Cleavage Sites (PICS), a mass spectrometry-based method utilizing database searchable proteome-derived peptide libraries. For each enzyme we identified > 100 unique-cleaved peptides, resulting in robust cleavage logos revealing collagen-like specificity patterns: a strong preference for glycine in P3 and P1′, proline at P2 and P2′, and a slightly looser specificity at P1, which in collagen is typically occupied by hydroxyproline. This specificity for the classic collagen motifs Gly-Pro-X and Gly-X-Hyp represents a remarkable adaptation considering the complex requirements for substrate unfolding and presentation that need to be fulfilled before a single collagen strand becomes accessible for cleavage.
Biological significance
We demonstrate the striking sequence specificity of a family of clostridial collagenases using proteome derived peptide libraries and PICS, Proteomic Identification of protease Cleavage Sites. In combination with the previously published crystal structures of these proteases, our results represent an important piece of the puzzle in understanding the complex mechanism underlying collagen hydrolysis, and pave the way for the rational design of specific test substrates and selective inhibitors.
This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?
Graphical abstract
•Active site specificity profiling of 3 clostridial collagenases—ColG and H from C. histolyticum, and ColT from C. tetani.•Their high sequence specificity to collagen-like sequence points towards a co-evolution with the mammalian substrate.•Significant differences to MMPs and a more promiscuous cleavage mechanism facilitating rapid collagenolysis were revealed.•Human proteome-derived peptide libraries & PICS are suitable for active site specificity profiling of pathogenic proteases.•Results pave the way for rational design of test substrates and selective inhibitors.
PMCID: PMC3985423  PMID: 24125730
Clostridia; Collagenase; MMPs; PICS; Mass spectrometry
9.  Nitration of the Birch Pollen Allergen Bet v 1.0101: Efficiency and Site-Selectivity of Liquid and Gaseous Nitrating Agents 
Journal of Proteome Research  2014;13(3):1570-1577.
Nitration 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.
PMCID: PMC3950889  PMID: 24517313
Bet v 1.0101; HPLC−MS/MS; tyrosine nitration; nitration sites; air pollution
10.  Activation of legumain involves proteolytic and conformational events, resulting in a context- and substrate-dependent activity profile 
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.
PMCID: PMC3253828  PMID: 22232165
cysteine proteases; pH regulation; conformational activation; substrate-dependent activity; autoimmune diseases; cancer
11.  Do-it-yourself histidine-tagged bovine enterokinase: A handy member of the protein engineer's toolbox☆ 
Journal of Biotechnology  2013;168(4):421-425.
•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.
PMCID: PMC3863954  PMID: 24184090
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
12.  Stabilization of the Dimeric Birch Pollen Allergen Bet v 1 Impacts Its Immunological Properties* 
The Journal of Biological Chemistry  2013;289(1):540-551.
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.
PMCID: PMC3879576  PMID: 24253036
Allergen; Crystal Structure; Mass Spectrometry (MS); Post-translational Modification; Protein Assembly; Dimerization; Noncanonical Amino Acid Incorporation; Polysulfide Linking; Position-specific Alteration of Genetic Code
13.  Crystal Structures of the Viral Protease Npro Imply Distinct Roles for the Catalytic Water in Catalysis 
Structure(London, England:1993)  2013;21(6):929-938.
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.
Graphical Abstract
•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.
PMCID: PMC3677099  PMID: 23643950
14.  Structural Basis for Activity Regulation and Substrate Preference of Clostridial Collagenases G, H, and T* 
The Journal of Biological Chemistry  2013;288(28):20184-20194.
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.
PMCID: PMC3711286  PMID: 23703618
Protease; Protein Degradation; Protein Structure; Proteolytic Enzymes; X-ray Crystallography; Collagenase; Metal Regulation
15.  Crystallographically Mapped Ligand Binding Differs in High and Low IgE Binding Isoforms of Birch Pollen Allergen Bet v 1 
Journal of Molecular Biology  2012;422(1):109-123.
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.
Graphical Abstract
► 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.
PMCID: PMC3422537  PMID: 22634284
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
16.  Structure of collagenase G reveals a chew and digest mechanism of bacterial collagenolysis 
Nature structural & molecular biology  2011;18(10):1109-1114.
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.
PMCID: PMC3191118  PMID: 21947205
17.  Real Space Refinement of Crystal Structures with Canonical Distributions of Electrons 
Structure(London, England:1993)  2011;19(12):1739-1743.
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
PMCID: PMC3234344  PMID: 22153496
18.  Natural and synthetic inhibitors of kallikrein-related peptidases (KLKs) 
Biochimie  2010;92(11):1546-1567.
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.
PMCID: PMC3014083  PMID: 20615447
Tissue kallikrein; Specificity pockets; Inhibitory compound; Zinc; Rule of five
19.  Crystallization and preliminary X-ray characterization of the catalytic domain of collagenase G from Clostridium histolyticum  
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%.
PMCID: PMC2376405  PMID: 18453715
collagenase G; Clostridium histolyticum
20.  A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli 
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.
PMCID: PMC3085789  PMID: 19333597
Clostridial collagenases; Expression; Purification; Platform

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