The dimerized pentraxin-like domain of the adhesion G protein-coupled receptor 112 (ADGRG4) suggests function in sensing mechanical forces.

Adhesion G protein-coupled receptors (aGPCRs) feature large extracellular regions with modular domains that often resemble protein classes of various function. The pentraxin (PTX) domain, which is predicted by sequence homology within the extracellular region of four different aGPCR members, is well known to form pentamers and other oligomers. Oligomerization of GPCRs is frequently reported and mainly driven by interactions of the seven-transmembrane region and N or C termini. While the functional importance of dimers is well-established for some class C GPCRs, relatively little is known about aGPCR multimerization. Here, we showcase the example of ADGRG4, an orphan aGPCR that possesses a PTX-like domain at its very N-terminal tip, followed by an extremely long stalk containing serine-threonine repeats. Using X-ray crystallography and biophysical methods, we determined the structure of this unusual PTX-like domain and provide experimental evidence for a homodimer equilibrium of this domain which is Ca2+-independent and driven by intermolecular contacts that differ vastly from the known soluble PTXs. The formation of this dimer seems to be conserved in mammalian ADGRG4 indicating functional relevance. Our data alongside of theoretical considerations lead to the hypothesis that ADGRG4 acts as an in vivo sensor for shear forces in enterochromaffin and Paneth cells of the small intestine.

Structural and Mechanistic Studies on Substrate and Stereoselectivity of the Indole Monooxygenase VpIndA1: New Avenues for Biocatalytic Epoxidations and Sulfoxidations.

Flavoprotein monooxygenases are a versatile group of enzymes for biocatalytic transformations. Among these, group E monooxygenases (GEMs) catalyze enantioselective epoxidation and sulfoxidation reactions. Here, we describe the crystal structure of an indole monooxygenase from the bacterium Variovorax paradoxus EPS, a GEM designated as VpIndA1. Complex structures with substrates reveal productive binding modes that, in conjunction with force-field calculations and rapid mixing kinetics, reveal the structural basis of substrate and stereoselectivity. Structure-based redesign of the substrate cavity yielded variants with new substrate selectivity (for sulfoxidation of benzyl phenyl sulfide) or with greatly enhanced stereoselectivity (from 35.1 % to 99.8 % ee for production of (1S,2R)-indene oxide). This first determination of the substrate binding mode of GEMs combined with structure-function relationships opens the door for structure-based design of these powerful biocatalysts.

Functional impact of intramolecular cleavage and dissociation of adhesion G protein-coupled receptor GPR133 (ADGRD1) on canonical signaling.

GPR133 (ADGRD1), an adhesion G protein-coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF-CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM.

Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity.

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.

Analysis of double-emulsion droplets with ESI mass spectrometry for monitoring lipase-catalyzed ester hydrolysis at nanoliter scale.

Microfluidic double-emulsion droplets allow the realization and study of biphasic chemical processes such as chemical reactions or extractions on the nanoliter scale. Double emulsions of the rare type (o1/w/o2) are used here to realize a lipase-catalyzed reaction in the non-polar phase. The surrounding aqueous phase induces the transfer of the hydrophilic product from the core oil phase, allowing on-the-fly MS analysis in single double droplets. A microfluidic two-step emulsification process is developed to generate the (o1/w/o2) double-emulsion droplets. In this first example of microfluidic double-emulsion MS coupling, we show in proof-of-concept experiments that the chemical composition of the water layer can be read online using ESI-MS. Double-emulsion droplets were further employed as two-phase micro-reactors for the hydrolysis of the lipophilic ester p-nitrophenyl palmitate catalyzed by the Candida antarctica lipase B (CalB). Finally, the formation of the hydrophilic reaction product p-nitrophenol within the double-emulsion droplet micro-reactors is verified by subjecting the double-emulsion droplets to online ESI-MS analysis.

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5'-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives.

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5'-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.

Ribosomal Target-Binding Sites of Antimicrobial Peptides Api137 and Onc112 Are Conserved among Pathogens Indicating New Lead Structures To Develop Novel Broad-Spectrum Antibiotics.

Proline-rich antimicrobial peptides expressed in insects are primarily active against Enterobacteriaceae. Mechanistically, they target the bacterial (70S) ribosome after partially transporter-based cellular uptake, as revealed for Api137 and Onc112 on Escherichia coli. Following molecular modeling indicating that the Onc112 contact site is conserved among the ribosomes of high-priority pathogens, the ribosome binding of Api137 and Onc112 was studied. The dissociation constants (Kd ) of Onc112 were ∼75 nmol/L for Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, 36 nmol/L for Pseudomonas aeruginosa, and 102 nmol/L for Staphylococcus aureus, thus indicating a very promising lead structure for developing broad-spectrum antibiotics. Api137 bound weaker with Kd values ranging from 155 nmol/L to 13 µmol/L. For most bacteria, the antibacterial activities were lower than predicted from the Kd values, which was only partially explained by their ability to enter bacterial cells. Other factors limiting the activity expected from the ribosome binding might be off-target binding.

Membrane Phospholipids and Polyphosphates as Cofactors and Binding Molecules of SERPINA12 (vaspin).

Visceral adipose tissue derived serine protease inhibitor (vaspin) is a member of the serpin family and has been shown to have beneficial effects on glucose tolerance, insulin stability as well as adipose tissue inflammation, parameters seriously affected by obesity. Some of these effects require inhibition of target proteases such as kallikrein 7(KLK7) and many studies have demonstrated vaspin-mediated activation of intracellular signaling cascades in various cells and tissues. So far, little is known about the exact mechanism how vaspin may trigger these intracellular signaling events. In this study, we investigated and characterized the interaction of vaspin with membrane lipids and polyphosphates as well as their potential regulatory effects on serpin activity using recombinant vaspin and KLK7 proteins and functional protein variants thereof. Here, we show for the first time that vaspin binds to phospholipids and polyphosphates with varying effects on KLK7 inhibition. Vaspin binds strongly to monophosphorylated phosphatidylinositol phosphates (PtdInsP) with no effect on vaspin activation. Microscale thermophoresis (MST) measurements revealed high-affinity binding to polyphosphate 45 (KD: 466 ± 75 nM) and activation of vaspin in a heparin-like manner. Furthermore, we identified additional residues in the heparin binding site in ß-sheet A by mutating five basic residues resulting in complete loss of high-affinity heparin binding. Finally, using lipid overlay assays, we show that these residues are additionally involved in PtdInsP binding. Phospholipids play a major role in membrane trafficking and signaling whereas polyphosphates are procoagulant and proinflammatory agents. The identification of phospholipids and polyphosphates as binding partners of vaspin will contribute to the understanding of vaspins involvement in membrane trafficking, signaling and beneficial effects associated with obesity.

Basic Residues of ß-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12).

Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules. In addition, basic residues contributing to heparin binding and heparin activation were identified by a selective labeling approach. Together, these data show that vaspin binds heparin with high affinity (KD = 21 ± 2 nm) and that binding takes place at a basic patch on top of ß-sheet A and is different from other heparin-binding serpins. Mutation of basic residues decreased heparin binding and activation of vaspin. Similarly, reactive center loop insertion into sheet A decreased heparin binding because it disturbs the basic cluster. Finally, using vaspin-overexpressing keratinocyte cells, we show that a significant part of secreted vaspin is bound in the extracellular matrix on the cell surface. Together, basic residues of central ß-sheet A contribute to heparin binding and activation of vaspin. Thus, binding to GAGs in the extracellular matrix can direct and regulate vaspin interaction with target proteases or other proteins and may play an important role in the various beneficial functions of vaspin in different tissues.

Kallikrein-related peptidase 14 is the second KLK protease targeted by the serpin vaspin.

Kallikrein-related peptidases KLK5, KLK7 and KLK14 are important proteases in skin desquamation and aberrant KLK activity is associated with inflammatory skin diseases such as Netherton syndrome but also with various serious forms of cancer. Previously, we have identified KLK7 as the first protease target of vaspin (Serpin A12). Here, we report KLK14 as a second KLK protease to be inhibited by vaspin. In conclusion, vaspin represents a multi-specific serpin targeting the kallikrein proteases KLK7 and KLK14, with distinct exosites regulating recognition of these target proteases and opposing effects of heparin binding on the inhibition reaction.

Glycosylation of human vaspin (SERPINA12) and its impact on serpin activity, heparin binding and thermal stability.

Vaspin is a glycoprotein with three predicted glycosylation sites at asparagine residues located in proximity to the reactive center loop and close to domains that play important roles in conformational changes underlying serpin function. In this study, we have investigated the glycosylation of human vaspin and its effects on biochemical properties relevant to vaspin function. We show that vaspin is modified at all three sites and biochemical data demonstrate that glycosylation does not hinder inhibition of the target protease kallikrein 7. Although binding affinity to heparin is slightly decreased, the protease inhibition reaction is still significantly accelerated in the presence of heparin. Glycosylation did not affect thermal stability.

Structural basis of the stereospecificity of bacterial B12-dependent 2-hydroxyisobutyryl-CoA mutase.

Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.

Crystal structure of cleaved vaspin (serpinA12).

The adipokine vaspin (serpinA12) is mainly expressed in white adipose tissue and exhibits various beneficial effects on obesity-related processes. Kallikrein 7 is the only known target protease of vaspin and is inhibited by the classical serpin inhibitory mechanism involving a cleavage of the reactive center loop between P1 (M378) and P1' (E379). Here, we present the X-ray structure of vaspin, cleaved between M378 and E379. We provide a comprehensive analysis of differences between the uncleaved and cleaved forms in the shutter, breach, and hinge regions with relation to common molecular features underlying the serpin inhibitory mode. Furthermore, we point out differences towards other serpins and provide novel data underlining the remarkable stability of vaspin. We speculate that the previously reported FKGx1Wx2x3 motif in the breach region may play a decisive role in determining the reactive center loop configuration in the native vaspin state and might contribute to the high thermostability of vaspin. Thus, this structure may provide a basis for future mutational studies.

Crystal structure of human platelet phosphofructokinase-1 locked in an activated conformation.

Phosphofructokinase-1 (Pfk) acts as the main control point of flux through glycolysis. It is involved in complex allosteric regulation and Pfk mutations have been linked to cancer development. Whereas the 3D structure and structural basis of allosteric regulation of prokaryotic Pfk has been studied in great detail, our knowledge about the molecular basis of the allosteric behaviour of the more complex mammalian Pfk is still very limited. To characterize the structural basis of allosteric regulation, the subunit interfaces and the functional consequences of modifications in Tarui's disease and cancer, we analysed the physiological homotetramer of human platelet Pfk at up to 2.67 Å resolution in two crystal forms. The crystallized enzyme is permanently activated by a deletion of the 22 C-terminal residues. Complex structures with ADP and fructose-6-phosphate (F6P) and with ATP suggest a role of three aspartates in the deprotonation of the OH-nucleophile of F6P and in the co-ordination of the catalytic magnesium ion. Changes at the dimer interface, including an asymmetry observed in both crystal forms, are the primary mechanism of allosteric regulation of Pfk by influencing the F6P-binding site. Whereas the nature of this conformational switch appears to be largely conserved in bacterial, yeast and mammalian Pfk, initiation of these changes differs significantly in eukaryotic Pfk.

A unique serpin P1' glutamate and a conserved ß-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin).

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1') of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1' glutamate (Glu(379)) with an arginine residue (Arg(302)) of the ß-sheet C. A P1' glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1' residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within ß-sheet C impeded the serpin-protease interaction. Arg(302) is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1' Glu(379), which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu(379) and Arg(302) influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.

Understanding the Structural Basis of Adhesion GPCR Functions.

Unlike conventional G-protein-coupled receptors (GPCRs), adhesion GPCRs (aGPCRs) have large extracellular regions that are autoproteolytically cleaved from their membrane-embedded seven-pass transmembrane helices. Autoproteolysis occurs within the conserved GPCR-Autoproteolysis INducing (GAIN) domain that is juxtaposed to the transmembrane domain and cleaves the last beta strand of the GAIN domain. The other domains of the extracellular region are variable and specific to each aGPCR and are likely involved in adhering to various ligands. Emerging evidence suggest that extracellular regions may modulate receptor function and that ligand binding to the extracellular regions may induce receptor activation via multiple mechanisms. Here, we summarize current knowledge about the structural understanding for the extracellular regions of aGPCRs and discuss their possible functional roles that emerge from the available structural information.

Crystal structure of NTPDase2 in complex with the sulfoanthraquinone inhibitor PSB-071.

In many vertebrate tissues CD39-like ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) act in concert with ecto-5'-nucleotidase (e5NT, CD73) to convert extracellular ATP to adenosine. Extracellular ATP is a cytotoxic, pro-inflammatory signalling molecule whereas its product adenosine constitutes a universal and potent immune suppressor. Interference with these ectonucleotidases by use of small molecule inhibitors or inhibitory antibodies appears to be an effective strategy to enhance anti-tumour immunity and suppress neoangiogenesis. Here we present the first crystal structures of an NTPDase catalytic ectodomain in complex with the Reactive Blue 2 (RB2)-derived inhibitor PSB-071. In both of the two crystal forms presented the inhibitor binds as a sandwich of two molecules at the nucleoside binding site. One of the molecules is well defined in its orientation. Specific hydrogen bonds are formed between the sulfonyl group and the nucleoside binding loop. The methylphenyl side chain functionality that improved NTPDase2-specificity is sandwiched between R245 and R394, the latter of which is exclusively found in NTPDase2. The second molecule exhibits great in-plane rotational freedom and could not be modelled in a specific orientation. In addition to this structural insight into NTPDase inhibition, the observation of the putative membrane interaction loop (MIL) in two different conformations related by a 10° rotation identifies the MIL as a dynamic section of NTPDases that is potentially involved in regulation of catalysis.

Structures of Legionella pneumophila NTPDase1 in complex with polyoxometallates.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are secreted or membrane-bound ectonucleotidases that hydrolyze the anhydride bonds of nucleoside triphosphates and nucleoside diphosphates. Mammalian cell-surface NTPDase enzymes are inhibited by various polyoxometallates. Here, the structures of NTPDase1 from the bacterium Legionella pneumophila (LpNTPDase1) in complex with the dodecatungstate POM-1, decavanadate and octamolybdate/heptamolybdate are described. The metal clusters are bound at different sites but always in a highly ordered fashion via electrostatic interactions and hydrogen bonds. For octamolybdate, covalent interactions after oxygen ligand exchange by a serine and histidine side chain are also observed. The potential inhibitory mechanism and the use of the metal clusters as phasing tools for new NTPDase structures are discussed. The binding mode of a tartrate ion at the catalytic centre suggests novel strategies for the structure-based design of NTPDase inhibitors, and the observation of the enzyme in an intermediate open state contributes to our understanding of NTPDase enzyme dynamics.

Crystal structure and catalytic mechanism of chloromuconolactone dehalogenase ClcF from Rhodococcus opacus 1CP.

The actinobacterium Rhodococcus opacus 1CP possesses a so far unique variant of the modified 3-oxoadipate pathway for 3-chlorocatechol degradation. One important feature is the novel dehalogenase ClcF, which converts (4R,5S)-5-chloromuconolactone to E-dienelactone. ClcF is related to muconolactone isomerase (MLI, EC 5.3.3.4). The enzyme has a ferredoxin-type fold and forms a homodecamer of 52-symmetry, typical for the MLI family. The active site is formed by residues from two monomers. The complex structure of an E27A variant with bound substrate in conjunction with mutational studies indicate that E27 acts as the proton acceptor in a univalent single-base syn-dehydrohalogenation mechanism. Despite the evolutionary specialization of ClcF, the conserved active-site structures suggest that the proposed mechanism is representative for the MLI family. Furthermore, ClcF represents a novel type of dehalogenase based on an isomerase scaffold.

Protein surface charge of trypsinogen changes its activation pattern.

BACKGROUND: Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein-protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein-protein interaction. RESULTS: Based on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme. CONCLUSION: The variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein-protein interactions, as shown here for human trypsinogen.