Calcium-induced structural transitions are central to the folding, function, and processing of serratiopeptidase zymogen into mature form.

Serratia marcescens is an emerging health-threatening, gram-negative opportunistic pathogen associated with a wide variety of localized and life-threatening systemic infections. One of the most crucial virulence factors produced by S. marcescens is serratiopeptidase, a 50.2-kDa repeats-in-toxin (RTX) family broad-specificity zinc metalloprotease. RTX family proteins are functionally diverse exoproteins of gram-negative bacteria that exhibit calcium-dependent structural dynamicity and are secreted through a common type-1 secretion system (T1SS) machinery. To evaluate the impact of various divalent ligands on the folding and maturation of serratiopeptidase zymogen, the protein was purified and a series of structural and functional investigations were undertaken. The results indicate that calcium binding to the C-terminal RTX domain acts as a folding switch, triggering a disordered-to-ordered transition in the enzyme's conformation. Further, the auto-processing of the 16-amino acid N-terminal pro-peptide results in the maturation of the enzyme. The binding of calcium ions to serratiopeptidase causes a highly cooperative conformational transition in its structure, which is essential for the enzyme's activation and maturation. This conformational change is accompanied by an increase in solubility and enzymatic activity. For efficient secretion and to minimize intracellular toxicity, the enzyme needs to be in an unfolded extended form. The calcium-rich extracellular environment favors the folding and processing of zymogen into mature serratiopeptidase, i.e., the holo-form required by S. marcescens to establish infections and survive in different environmental niches.

A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection.

Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.

In silico insights into procathepsin S maturation mediated by glycosaminoglycans.

Procathepsins, inactive precursors of cathepsins are present in the extracellular matrix (ECM) and in lysosomes. Their active forms are involved in a number of biologically relevant processes, including bone resorption, intracellular proteolysis and regulation of programmed cell death. These processes might be mediated by glycosaminoglycans (GAGs), long unbranched periodic negatively charged polysaccharides. GAGs are also present in ECM and play important role in anticoagulation, angiogenesis and tissue regeneration. GAGs not only mediate the enzymatic activity of cathepsins but can also regulate the process of procathepsin maturation, as it was shown for procathepsin B and S. In this study, we propose the molecular mechanism underlying the biological role of GAGs in procathepsin S maturation and compare our findings with computational data obtained for procathepsin B. We rigorously analyse procathepsin S-GAG complexes in terms of their dynamics, free energy and potential allosteric regulation. We conclude that the GAG binding region might have an effect on the dynamics of procathepsin S structure and so affect its maturation by two different mechanisms.

Factor XII Explored with AlphaFold - Opportunities for Selective Drug Development.

Medical device associated thrombosis is an important clinical problem. This type of thrombosis can result from Factor XII (FXII) binding to non-natural surface materials and subsequent activation of the contact pathway. This drives the development of new therapeutic strategies to block this pathway and information on the structural properties of FXII should catalyse this quest. Presently, there is no publicly available crystal structure of full-length FXII. However, the AlphaFold Protein Structure Database provides a model structure. We here explore this model in combination with previous structure-function studies to identify opportunities for selective pharmacological blockade of the contribution of FXII in medical device associated thrombosis. Previous studies demonstrated that FXII activation is dependent on molecular cleavage after R353. We subsequently proposed that protein conformation protects this cleavage site to ensure zymogen quiescence and prevent inappropriate FXII activation. The AlphaFold model shows that a small loop containing R353 indeed is buried in the globular molecule. This is the result of intra-molecular interactions between the (N-terminal) Fibronectin type II domain, (central) kringle and (C-terminal) protease domain, in a structure that resembles a three-point harness. Furthermore, this interaction pushes the intermediate domains, as well as the flexible proline-rich region (PRR), outward while encapsulating R353 in the molecule. The outward directed positively charged patches are likely to be involved in binding to anionic surfaces. The binding of FXII to surfaces (and several monoclonal antibodies) acccelerates its activation by inducing conformational changes. For prevention of medical device associated thrombosis, it is therefore important to target the surface binding sites of FXII without causing structural changes.

Modifying a bacterial tyrosinase zymogen for use in protease activity assays.

Current clinical laboratory assays are not sufficient for determining the activity of many specific human proteases yet. In this study, we developed a general approach that enables the determination of activities of caspase-3 based on the proteolytic activation of the engineered zymogen of the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase) by detecting the diphenolase activity in an increase in absorbance at 475 nm. Here, we designed three different zymogen constructs of Vs-tyrosinase, including RSL-pre-pro-TYR, Pre-pro-TYR, and Pro-TYR. The active domain was fused to the reactive site loop (RSL) of α1-proteinase inhibitor and/or its own signal peptide (pre) and/or its own C-terminal domain (pro) via a linker containing a specific caspase-3 cleavage site. Further studies revealed that both RSL peptide and TAT signal peptide were able to inhibit tyrosinase diphenolase activity, in which RSL-pre-pro-TYR had the lowest background signals. Therefore, a specific protease activity such as caspase-3 could be detected when a suitable zymogen was established. Our results could provide a new way to directly detect the activities of key human proteases, for instance, to monitor the efficacy and safety of tumor therapy by determining the activity of apoptosis-related caspase-3 in patients. KEY POINTS: • RSL inhibited the activity of Verrucomicrobium spinosum tyrosinase. • N-pre and C-terminal domain exerted stronger dual inhibition on the Vs-tyrosinase. • The activity of caspase-3 could be measured by the zymogen activation system.

Zymogenic latency in an ∼250-million-year-old astacin metallopeptidase.

The horseshoe crab Limulus polyphemus is one of few extant Limulus species, which date back to ∼250 million years ago under the conservation of a common Bauplan documented by fossil records. It possesses the only proteolytic blood-coagulation and innate immunity system outside vertebrates and is a model organism for the study of the evolution and function of peptidases. The astacins are a family of metallopeptidases that share a central ∼200-residue catalytic domain (CD), which is found in >1000 species across holozoans and, sporadically, bacteria. Here, the zymogen of an astacin from L. polyphemus was crystallized and its structure was solved. A 34-residue, mostly unstructured pro-peptide (PP) traverses, and thus blocks, the active-site cleft of the CD in the opposite direction to a substrate. A central `PP motif' (F35-E-G-D-I39) adopts a loop structure which positions Asp38 to bind the catalytic metal, replacing the solvent molecule required for catalysis in the mature enzyme according to an `aspartate-switch' mechanism. Maturation cleavage of the PP liberates the cleft and causes the rearrangement of an `activation segment'. Moreover, the mature N-terminus is repositioned to penetrate the CD moiety and is anchored to a buried `family-specific' glutamate. Overall, this mechanism of latency is reminiscent of that of the other three astacins with known zymogenic and mature structures, namely crayfish astacin, human meprin ß and bacterial myroilysin, but each shows specific structural characteristics. Remarkably, myroilysin lacks the PP motif and employs a cysteine instead of the aspartate to block the catalytic metal.

Chemical zymogens for the protein cysteinome.

We present three classes of chemical zymogens established around the protein cysteinome. In each case, the cysteine thiol group was converted into a mixed disulfide: with a small molecule, a non-degradable polymer, or with a fast-depolymerizing fuse polymer (ZLA). The latter was a polydisulfide based on naturally occurring molecule, lipoic acid. Zymogen designs were applied to cysteine proteases and a kinase. In each case, enzymatic activity was successfully masked in full and reactivated by small molecule reducing agents. However, only ZLA could be reactivated by protein activators, demonstrating that the macromolecular fuse escapes the steric bulk created by the protein globule, collects activation signal in solution, and relays it to the active site of the enzyme. This afforded first-in-class chemical zymogens that are activated via protein-protein interactions. We also document zymogen exchange reactions whereby the polydisulfide is transferred between the interacting proteins via the "chain transfer" bioconjugation mechanism.

Massively parallel, computationally guided design of a proenzyme.

Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.

Structures of plasmepsin X from Plasmodium falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme.

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

Structural characterisation of inhibitory and non-inhibitory MMP-9-TIMP-1 complexes and implications for regulatory mechanisms of MMP-9.

MMP-9 plays a number of important physiological functions but is also responsible for many pathological processes, including cancer invasion, metastasis, and angiogenesis. It is, therefore, crucial to understand its enzymatic activity, including activation and inhibition mechanisms. This enzyme may also be partially involved in the "cytokine storm" that is characteristic of COVID-19 disease (SARS-CoV-2), as well as in the molecular mechanisms responsible for lung fibrosis. Due to the variety of processing pathways involving MMP-9 in biological systems and its uniqueness due to the O-glycosylated domain (OGD) and fibronectin-like (FBN) domain, specific interactions with its natural TIMP-1 inhibitor should be carefully studied, because they differ significantly from other homologous systems. In particular, earlier experimental studies have indicated that the newly characterised circular form of a proMMP-9 homotrimer exhibits stronger binding properties to TIMP-1 compared to its monomeric form. However, molecular structures of the complexes and the binding mechanisms remain unknown. The purpose of this study is to fill in the gaps in knowledge. Molecular modelling methods are applied to build the inhibitory and non-inhibitory MMP-9-TIMP-1 complexes, which allows for a detailed description of these structures and should allow for a better understanding of the regulatory processes in which MMP-9 is involved.

Oxygen-producing proenzyme hydrogels for photodynamic-mediated metastasis-inhibiting combinational therapy.

Photodynamic therapy (PDT) has provided a promising approach for the treatment of solid tumors, while the therapeutic efficacy is often limited due to the hypoxic tumor microenvironment, resulting in tumor metastasis. Herein, we report an oxygen-producing proenzyme hydrogel (OPeH) with photoactivatable enzymatic activity for PDT enabled metastasis-inhibiting combinational therapy of breast cancer. This OPeH based on alginate is composed of protoporphyrin IX (PpIX) conjugated manganese oxide (MnO2) nanoparticles, which act as both the photosensitizer and oxygen-producing agent, and singlet oxygen (1O2)-responsive proenzyme nanoparticles. In the hypoxic and acidic tumor microenvironment, MnO2 can generate 1O2 to promote PpIX-mediated PDT with an amplified 1O2 generation efficiency, which also triggers the cleavage of 1O2-responsive linkers and cascade activation of proenzymes for cancer cell death. This combinational therapy upon photoactivation not only greatly inhibited the tumor growth, but also suppressed lung metastasis in a mouse xenograft breast tumor model, which is impossible in the case of PDT alone. This study thus provides a proenzyme hydrogel platform with photoactivatable activity for metastasis-inhibiting cancer therapy with high efficacy and safety.

The role of propeptide-mediated autoinhibition and intermolecular chaperone in the maturation of cognate catalytic domain in leucine aminopeptidase.

Leucyl aminopeptidase A from Aspergillus oryzae RIB40 (AO-LapA) is an exo-acting peptidase, widely utilised in food debittering applications. AO-LapA is secreted as a zymogen by the host and requires enzymatic cleavage of the autoinhibitory propeptide to reveal its full activity. Scarcity of structural data of zymogen aminopeptidases hampers a better understanding of the details of their molecular action of autoinhibition and how this might be utilised to improve the properties of such enzymes by recombinant methods for more effective bioprocessing. To address this gap in the literature, herein we report high-resolution crystal structures of recombinantly expressed AO-LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and AO-mLapA complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from Pichia pastoris culture supernatant. Our structures reveal a plausible molecular mechanism of LapA catalytic domain autoinhibition by propeptide and highlights the role of intramolecular chaperone (IMC). Our data suggest an absolute requirement for IMC in the maturation of cognate catalytic domain of AO-LapA. This observation is reinforced by our expression and refolding data of catalytic domain only (AO-refLapA) from Escherichia coli inclusion bodies, revealing a limited active conformation. Our work supports the notion that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and corresponding catalytic domain, demonstrated in our AO-proLapA zymogen crystal structure. Furthermore, understanding the atomic details of the autoinhibitory mechanism of cognate catalytic domains by native propeptides has wider reaching implications toward synthetic production of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes that share an analogous reaction mechanism.

Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes.

Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.

Structure of the Plasmodium falciparum PfSERA5 pseudo-zymogen.

PfSERA5, a significantly abundant protein present within the parasitophorous vacuole (PV) and essential for normal growth during the blood-stage life cycle of the malaria parasite Plasmodium falciparum, displays structural similarity to many other cysteine proteases. However, PfSERA5 does not exhibit any detectable protease activity and therefore the role of the PfSERA5 papain-like domain (PfSERA5E), thought to remain bound to its cognate prodomain, remains unknown. In this study, we present a revised structure of the central PfSERA5E domain at a resolution of 1.2 Å, and the first structure of the "zymogen" of this papain-like domain including its cognate prodomain (PfSERA5PE) to 2.2 Å resolution. PfSERA5PE is somewhat structurally similar to that of other known proenzymes, retaining the conserved overall folding and orientation of the prodomain through, and occluding, the archetypal papain-like catalytic triad "active-site" cleft, in the same reverse direction as conventional prodomains. Our findings are congruent with previously identified structures of PfSERA5E and of similar "zymogens" and provide a foundation for further investigation into the function of PfSERA5.

Zymogen and activated protein C have similar structural architecture.

Activated protein C is a trypsin-like protease with anticoagulant and cytoprotective properties that is generated by thrombin from the zymogen precursor protein C in a reaction greatly accelerated by the cofactor thrombomodulin. The molecular details of this activation remain elusive due to the lack of structural information. We now fill this gap by providing information on the overall structural organization of these proteins using single molecule FRET and small angle X-ray scattering. Under physiological conditions, both zymogen and protease adopt a conformation with all domains vertically aligned along an axis 76 Å long and maximal particle size of 120 Å. This conformation is stabilized by binding of Ca2+ to the Gla domain and is affected minimally by interaction with thrombin. Hence, the zymogen protein C likely interacts with the thrombin-thrombomodulin complex through a rigid body association that produces a protease with essentially the same structural architecture. This scenario stands in contrast to an analogous reaction in the coagulation cascade where conversion of the zymogen prothrombin to the protease meizothrombin by the prothrombinase complex is linked to a large conformational transition of the entire protein. The presence of rigid epidermal growth factor domains in protein C as opposed to kringles in prothrombin likely accounts for the different conformational plasticity of the two zymogens. The new structural features reported here for protein C have general relevance to vitamin K-dependent clotting factors containing epidermal growth factor domains, such as factors VII, IX, and X.

Mild acidity likely accelerates the physiological matriptase autoactivation process: a comparative study between spontaneous and acid-induced matriptase zymogen activation.

The pathophysiological functions of matriptase, a type 2 transmembrane serine protease, rely primarily on its enzymatic activity, which is under tight control through multiple mechanisms. Among those regulatory mechanisms, the control of zymogen activation is arguably the most important. Matriptase zymogen activation not only generates the mature active enzyme but also initiates suppressive mechanisms, such as rapid inhibition by HAI-1, and matriptase shedding. These tightly coupled events allow the potent matriptase tryptic activity to fulfill its biological functions at the same time as limiting undesired hazards. Matriptase is converted to the active enzyme via a process of autoactivation, in which the activational cleavage is thought to rely on the interactions of matriptase zymogen molecules and other as yet identified proteins. Matriptase autoactivation can occur spontaneously and is rapidly followed by the formation and then shedding of matriptase-HAI-1 complexes, resulting in the presence of relatively low levels of the complex on cells. Activation can also be induced by several non-protease factors, such as the exposure of cells to a mildly acidic buffer, which rapidly causes high-level matriptase zymogen activation in almost all cell lines tested. In the current study, the structural requirements for this acid-induced zymogen activation are compared with those required for spontaneous activation through a systematic analysis of the impact of 18 different mutations in various structural domains and motifs on matriptase zymogen activation. Our study reveals that both acid-induced matriptase activation and spontaneous activation depend on the maintenance of the structural integrity of the serine protease domain, non-catalytic domains, and posttranslational modifications. The common requirements of both modes of activation suggest that acid-induced matriptase activation may function as a physiological mechanism to induce pericellular proteolysis by accelerating matriptase autoactivation.

Recombinant production of active microbial transglutaminase in E. coli by using self-cleavable zymogen with mutated propeptide.

Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7 ± 2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.

Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review.

Endotoxin testing by recombinant factor C (rFC) is increasing with the addition of new suppliers of reagents. By use of a recombinantly produced factor C , based on the sequence of a coagulation enzyme present in horseshoe crab amebocyte lysates, the rFC tests are designed as substitutes for the traditional Limulus amebocyte lysate (LAL)/Tachypleus amebocyte lysate tests based on horseshoe crab blood. Comparative testing of samples with both the LAL and recombinant reagents has shown a high degree of correlation, suggesting that use of rFC is comparable to the more traditional LAL tests and may be technologically superior. Recombinant factor C does not recognize the factor G pathway, the alternate coagulation pathway that the lysate reagents detect. This feature allows rFC to detect endotoxin more selectively. As a recombinantly produced material, it avoids the use of the horseshoe crabs required for lysate production, thereby protecting this species, which is at risk in some parts of the world. Recombinant factor C is expected to further benefit from a more sustainable supply chain based upon a robust biotechnological production process. We summarize here the results of many studies that evaluated the use of recombinant technology for the detection of environmental endotoxin. Additionally, we include a review of the current compendia and regulatory status of the recombinant technologies for use in the quality control of pharmaceutical manufacturing. Our analysis confirms that the recombinant technologies are comparable in protecting patient safety.

Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function.

One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin ß-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO- had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into ß-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles.

Roles of the clip domains of two protease zymogens in the coagulation cascade in horseshoe crabs.

The lipopolysaccharide (LPS)-triggered coagulation cascade in horseshoe crabs comprises three protease zymogens: prochelicerase C (proC), prochelicerase B (proB), and the proclotting enzyme (proCE). The presence of LPS results in autocatalytic activation of proC to α-chelicerase C, which, in turn, activates proB to chelicerase B, converting proCE to the clotting enzyme (CE). ProB and proCE contain an N-terminal clip domain, but the roles of these domains in this coagulation cascade remain unknown. Here, using recombinant proteins and kinetics and binding assays, we found that five basic residues in the clip domain of proB are required to maintain its LPS-binding activity and activation by α-chelicerase C. Moreover, an amino acid substitution at a potential hydrophobic cavity in proB's clip domain (V55A-proB) reduced both its LPS-binding activity and activation rate. WT proCE exhibited no LPS-binding activity, and the WT chelicerase B-mediated activation of a proCE variant with a substitution at a potential hydrophobic cavity (V53A-proCE) was ∼4-fold slower than that of WT proCE. The kcat/Km value of the interaction of WT chelicerase B with V53A-proCE was 7-fold lower than that of the WT chelicerase B-WT proCE interaction. The enzymatic activities of V55A-chelicerase B and V53A-CE against specific peptide substrates were indistinguishable from those of the corresponding WT proteases. In conclusion, the clip domain of proB recruits it to a reaction center composed of α-chelicerase C and LPS, where α-chelicerase C is ready to activate proB, leading to chelicerase B-mediated activation of proCE via its clip domain.