Antibacterial and Antifouling Properties of the Horseshoe Crab Carcinoscorpius rotundicauda.

<b>Background and Objective:</b> Horseshoe crabs are widely used in both traditional and modern pharmaceutical applications. Most of the previous studies on horseshoe crabs focused on their blood which contains hemolymph and amoebocyte lysate. This study aimed to determine the potential antibacterial and antifouling properties of different extracts from the carapace and the book gills of <i>Carcinoscorpius rotundicauda</i>. <b>Materials and Methods:</b> The crude extracts were subjected to the bioactivity tests using the disc-diffusion and the inhibition of biofilm-formation measurement assays, for both the antibacterial and antifouling activities respectively. <b>Results:</b> The results obtained indicated that the carapace extracts had stronger antibacterial and antifouling effects compared to the book gills extracts. Extracts obtained from the male displayed more activity compared to the extracts from the female with a few exceptions. Methanol and acetone carapace crude extracts showed the best overall performance. A sterol compound was isolated from the carapace acetone extracts of the male of <i>C. rotundicauda</i>. However, the compound did not display strong activity compared to the crude extract. The compound might be contributing to the observed activity with other components through a synergistic effect. <b>Conclusion:</b> The presence of antibacterial and antifouling activities in the carapace and book gills extracts could be added to the complexity of the defence mechanisms of horseshoe crabs. The results of this study, therefore, may contribute to the knowledge of the defence mechanisms of <i>C. rotundicauda</i>. Further research is needed to determine the bioactivities of other parts of the animal and to explore their potential applications.

A mutant equipped with a regenerated disulphide for the missing His loop of a serine protease zymogen in the horseshoe crab coagulation cascade.

The lipopolysaccharide (LPS)-triggered coagulation cascade in horseshoe crabs is composed of three zymogens belonging to the trypsinogen family: prochelicerase C, prochelicerase B (proB) and the proclotting enzyme (proCE). Trypsinogen-family members contain three conserved disulphides located around the active site. While it is known that proB evolutionarily lost one of the disulphides, the His-loop disulphide, the roles of the missing His-loop disulphide in proB remain unknown. Here, we prepared a proB mutant, named proB-murasame, equipped with a regenerated His-loop disulphide. The activation rate by upstream α-chelicerase C for proB-murasame was indistinguishable from that for wild-type (WT) proB. The resulting protease chelicerase B-murasame exhibited an 8-fold higher kcat value for downstream proCE than WT chelicerase B, whereas the Km value of chelicerase B-murasame was equivalent to that of WT chelicerase B. WT serpins-1, -2 and -3, identified as scavengers for the cascade, had no reactivity against WT chelicerase B, whereas chelicerase B-murasame was inhibited by WT serpin-2, suggesting that WT chelicerae B may trigger as-yet-unsolved phenomena after performing its duty in the cascade. The reconstituted LPS-triggered cascade containing proB-murasame exhibited ∼5-fold higher CE production than that containing WT proB. ProB-murasame might be used as a high value-adding reagent for LPS detection.

A highly sensitive endotoxin sensor based on redox cycling in a nanocavity.

We report a highly sensitive and rapid electrochemical method for the detection of endotoxin, based on a Limulus amebocyte lysate (LAL) assay using redox cycling at a pair of electrodes in a nanocavity for electrochemical signal amplification. We have previously developed Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as a substrate for the amperometric LAL assay, and in this work, Z-Leu-Gly-Arg-aminomethylferrocene (LGR-AMF) was newly prepared. They were examined as substrates for a LAL-based endotoxin assay using a nanocavity device. During the last step of the endotoxin-induced LAL cascade reaction, pAP or AMF is generated from the substrate, which can be detected electrochemically with efficient signal amplification by redox cycling between the two electrodes in the nanocavity. A device with a 190 nm-high nanocavity was fabricated by photolithography. With the fabricated device in model assay solutions prepared by mixing LGR-pAP and pAP, we demonstrated that pAP could be quantitatively detected from the difference in oxidation potentials between LGR-pAP and pAP. For LGR-AMF and AMF, a difference in the formal potential of 0.1 V was obtained which was considered to be insufficient to distinguish AMF from LGR-AMF. However, we showed for the first time that analytes such as AMF can be detected by differences in diffusion coefficients between the analyte and coexisting molecules (such as LGR-AMF) using a device with high redox-cycling efficiency. Next, the endotoxin assay was performed using the fabricated nanocavity device. Using this method, endotoxin was detected at concentrations as low as 0.2 and 0.5 EU L-1 after LAL reaction times of 1 h and 30 min, respectively, using the LGR-pAP substrate. However, the endotoxin assay using LGR-AMF was not successful because the clotting enzyme did not react with LGR-AMF. This problem might be solved by further design of the substrate. Our nanocavity device represents an effective platform for the simple and rapid detection of endotoxin with high sensitivity.

Intermolecular autocatalytic activation of serine protease zymogen factor C through an active transition state responding to lipopolysaccharide.

Horseshoe crab hemolymph coagulation is believed to be triggered by the autocatalytic activation of serine protease zymogen factor C to the active form, α-factor C, belonging to the trypsin family, through an active transition state of factor C responding to bacterial lipopolysaccharide (LPS), designated factor C*. However, the existence of factor C* is only speculative, and its proteolytic activity has not been validated. In addition, it remains unclear whether the proteolytic cleavage of the Phe737-Ile738 bond (Phe737 site) of factor C required for the conversion to α-factor C occurs intramolecularly or intermolecularly between the factor C molecules. Here we show that the Phe737 site of a catalytic Ser-deficient mutant of factor C is LPS-dependently hydrolyzed by a Phe737 site-uncleavable mutant, clearly indicating the existence of the active transition state of factor C without cleavage of the Phe737 site. Moreover, we found the following facts using several mutants of factor C: the autocatalytic cleavage of factor C occurs intermolecularly between factor C* molecules on the LPS surface; factor C* does not exhibit intrinsic chymotryptic activity against the Phe737 site, but it may recognize a three-dimensional structure around the cleavage site; and LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C* by interacting with the N-terminal region but also to allow the Phe737 site to be cleaved by inducing a conformational change around the Phe737 site or by acting as a scaffold to induce specific protein-protein interactions between factor C* molecules.

Ammonia excretion and acid-base regulation in the American horseshoe crab, Limulus polyphemus.

Many studies have investigated ammonia excretion and acid-base regulation in aquatic arthropods, yet current knowledge of marine chelicerates is non-existent. In American horseshoe crabs (Limulus polyphemus), book gills bear physiologically distinct regions: dorsal and ventral half-lamellae, a central mitochondria-rich area (CMRA) and peripheral mitochondria-poor areas (PMPAs). In the present study, the CMRA and ventral half-lamella exhibited characteristics important for ammonia excretion and/or acid-base regulation, as supported by high expression levels of Rhesus-protein 1 (LpRh-1), cytoplasmic carbonic anhydrase (CA-2) and hyperpolarization-activated cyclic nucleotide-gated K+ channel (HCN) compared with the PMPA and dorsal half-lamella. The half-lamellae displayed remarkable differences; the ventral epithelium was ion-leaky whereas the dorsal counterpart possessed an exceptionally tight epithelium. LpRh-1 was more abundant than Rhesus-protein 2 (LpRh-2) in all investigated tissues, but LpRh-2 was more prevalent in the PMPA than in the CMRA. Ammonia influx associated with high ambient ammonia (HAA) treatment was counteracted by intact animals and complemented by upregulation of branchial CA-2, V-type H+-ATPase (HAT), HCN and LpRh-1 mRNA expression. The dorsal epithelium demonstrated characteristics of active ammonia excretion. However, an influx was observed across the ventral epithelium as a result of the tissue's high ion conductance, although the influx rate was not proportionately high considering the ∼3-fold inwardly directed ammonia gradient. These novel findings suggest a role for the coxal gland in excretion and in the maintenance of hemolymph ammonia regulation under HAA. Hypercapnic exposure induced compensatory respiratory acidosis and partial metabolic depression. Functional differences between the two halves of a branchial lamella may be physiologically beneficial in reducing the backflow of waste products into adjacent lamellae, especially in fluctuating environments where ammonia levels can increase.

The Michaelis Complex of Arginine Kinase Samples the Transition State at a Frequency That Matches the Catalytic Rate.

Arginine kinase (AK), which is a member of the phosphagen kinase family, serves as a model system for studying the structural and dynamic determinants of biomolecular enzyme catalysis of all major states involved of the enzymatic cycle. These states are the apo state (substrate free), the Michaelis complex analogue AK:Arg:Mg·AMPPNP (MCA), a product complex analogue AK:pAIE:Mg·ADP (PCA), and the transition state analogue AK:Arg:Mg·ADP:NO3- (TSA). The conformational dynamics of these states have been studied by NMR relaxation dispersion measurements of the methyl groups of the Ile, Leu, and Val residues at two static magnetic fields. Although all states undergo significant amounts of µs-ms time scale dynamics, only the MCA samples a dominant excited state that resembles the TSA, as evidenced by the strong correlation between the relaxation dispersion derived chemical shift differences Δω and the equilibrium chemical shift differences Δδ of these states. The average lifetime of the MCA is 36 ms and the free energy difference to the TSA-like form is 8.5 kJ/mol. It is shown that the conformational energy landscape of the Michaelis complex analogue is shaped in a way that at room temperature it channels passage to the transition state, thereby determining the rate-limiting step of the phosphorylation reaction of arginine. Conversely, relaxation dispersion experiments of the TSA reveal that it samples the structures of the Michaelis complex analogue or the apo state as its dominant excited state. This reciprocal behavior shows that the free energy of the TSA, with all ligands bound, is lower by only about 8.9 kJ/mol than that of the Michaelis or apo complex conformations with the TSA ligands present.

The Sampling of Conformational Dynamics in Ambient-Temperature Crystal Structures of Arginine Kinase.

Arginine kinase provides a model for functional dynamics, studied through crystallography, enzymology, and nuclear magnetic resonance. Structures are now solved, at ambient temperature, for the transition state analog (TSA) complex. Analysis of quasi-rigid sub-domain displacements show that differences between the two TSA structures average about 5% of changes between substrate-free and TSA forms, and they are nearly co-linear. Small backbone hinge rotations map to sites that also flex on substrate binding. Anisotropic atomic displacement parameters (ADPs) are refined using rigid-body TLS constraints. Consistency between crystal forms shows that they reflect intrinsic molecular properties more than crystal lattice effects. In many regions, the favored directions of thermal/static displacement are appreciably correlated with movements on substrate binding. Correlation between ADPs and larger substrate-associated movements implies that the latter approximately follow paths of low-energy intrinsic motions.

Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade.

Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to ß-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by ß-factor C, in an LPS-dependent manner and that ß-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C.

The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.

Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.

A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation.

Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.

Phenoloxidase activity and thermostability of Cancer pagurus and Limulus polyphemus hemocyanin.

The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (K(m) value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while k(cat) is highest for catechol (2.44 min(-1) versus 0.67 min(-1) for l-Dopa and 0.71 min(-1) for dopamine). On treatment with 4mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T(m)~80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T(m)~92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.

Crystal structures of arginine kinase in complex with ADP, nitrate, and various phosphagen analogs.

Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and l-arginine and is a monomeric homolog of the human enzyme creatine kinase. Arginine and creatine kinases belongs to the phosphagen kinase family of enzymes, which consists of eight known members, each of which is specific for its own phosphagen. Here, the source of phosphagen specificity in arginine kinase is investigated through the use of phosphagen analogs. Crystal structures have been determined for Limulus polyphemus arginine kinase with one of four arginine analogs bound in a transition state analog complex: l-ornithine, l-citrulline, imino-l-ornithine, and d-arginine. In all complexes, the enzyme achieves a closed conformation very similar to that of the cognate transition state analog complex, but differences are observed in the configurations of bound ligands. Arginine kinase exhibits no detectable activity towards ornithine, citrulline, or imino-l-ornithine, and only trace activity towards d-arginine. The crystal structures presented here demonstrate that phosphagen specificity is derived neither from a lock-and-key mechanism nor a modulation of induced-fit conformational changes, but potentially from subtle distortions in bound substrate configurations.

Effects of known phenoloxidase inhibitors on hemocyanin-derived phenoloxidase from Limulus polyphemus.

Inhibitors of phenoloxidase are used routinely to characterise the structural and functional properties of phenoloxidases. Hemocyanin-derived phenoloxidase activity is also sensitive to standard phenoloxidase inhibitors. In this study, we characterise the effects of a number of phenoloxidase inhibitors on hemocyanin-derived phenoloxidase activity from the chelicerate, Limulus polyphemus. Both inhibition type and K(i) values were similar to those observed for hemocyanin-derived phenoloxidase from another chelicerate, Eurypelma californicum. In addition, substrate inhibition was observed at concentrations above 2mM dopamine. The conformation in which two of the inhibitors, namely tropolone and kojic acid, would bind near the Cu(II) centre of hemocyanin is proposed.

Simplified preparation of crude and functional coagulogen by thermal inactivation of serine proteases in Limulus amebocyte lysate and its application for rapid endotoxin determination.

The effects of thermal treatment on Limulus amebocyte lysate (LAL) reagent were studied. Thermal resistances of enzymes and coagulogen in LAL reagent were evaluated by aggregometry and SDS-PAGE. Although enzyme activities of LAL reagent were completely lost after heating at temperatures above 60 °C for 10 min, gelating activities of coagulogen were retained even over 80 °C. Phenylmethanesulfonyl fluoride (PMSF; 1 mmol/mL), a strong non-specific serine-protease inhibitor, did not completely inactivate serine-protease activities of LAL. As a result, complete hydrolysis of coagulogen to coagulin was unexpectedly obtained. Solvent treatment of LAL was similar in effect to thermal treatment of LAL, but there were 2 problems: complete removal of solvent from samples and increased solution turbidity during preparation. To study the application of thermal-treated LAL, we conjugated it with titania particles. LAL-conjugated titania particles were obtained as small aggregates between titania nanoparticles and thermal-treated LAL (LAL-conjugated microbeads; LCM). When the mixture of LCMs and fresh LAL reagent was reacted with endotoxin an acute aggregation of LCMs was induced prior to the aggregate formation of LAL as monitored by stirring turbidimetry. This method, endotoxin microbeads aggregometry (EMA) may provide a rapid and sensitive method for endotoxin determination.

Structural basis for dual-inhibition mechanism of a non-classical Kazal-type serine protease inhibitor from horseshoe crab in complex with subtilisin.

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Šresolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.

Rate-limiting domain and loop motions in arginine kinase.

Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and arginine. It is the arthropod homologue of creatine kinase, buffering cellular ATP levels. Crystal structures of arginine kinase, in substrate-free and substrate-bound forms, have revealed large conformational changes associated with the catalytic cycle. Recent nuclear magnetic resonance identified movements of the N-terminal domain and a loop comprising residues I182--G209 with conformational exchange rates in the substrate-free enzyme similar to the turnover rate. Here, to understand whether these motions might be rate-limiting, we determined activation barriers for both the intrinsic dynamics and enzyme turnover using measurements over a temperature range of 15-30 °C. (15)N transverse relaxation dispersion yields activation barriers of 46 ± 8 and 34 ± 12 kJ/mol for the N-terminal domain and I182--G209 loop, respectively. An activation barrier of 34 ± 13 kJ/mol was obtained for enzyme turnover from steady-state kinetics. The similarity between the activation barriers is indeed consistent with turnover being limited by backbone conformational dynamics and pinpoints the locations of potentially rate-limiting motions.

Allozyme polymorphisms in horseshoe crabs, Carcinoscorpius rotundicauda, collected from polluted and unpolluted intertidal areas in Peninsular Malaysia.

It has been widely reported that allozyme frequency variation is a potential indicator of heavy metal-induced impacts in aquatic populations. In the present study, wild populations of horseshoe crab (Carcinoscorpius rotundicauda) were collected from contaminated and uncontaminated sites of Peninsular Malaysia. By adopting horizontal starch gel electrophoresis, seven enzyme systems were used to study allozyme polymorphisms. Nine polymorphic loci were observed in C. rotundicauda. The relationships of allozyme variations with the concentrations of Cd, Cu, Ni, and Zn in sediments and in muscle tissues of horseshoe crabs were determined. Based on genetic distance, the lower mean value of Nei's D (0.017) indicated that both of the contaminated populations of Kg. Pasir Puteh and Kuala Juru were very closely related when compared to the relatively uncontaminated Pantai Lido population. Higher heterozygosities were shown by the contaminated populations when compared to the uncontaminated population. Different allelic frequencies could be observed for the aldolase (ALD; E.C. 2.7.5.1) locus between the contaminated and uncontaminated populations of C. rotundicauda. The dendrogram of genetic relationships of the three populations of C. rotundicauda showed the same clustering pattern as the dendrograms are based on heavy metals in the sediments and in the horseshoe crabs' abdominal muscles. From the F statistics, the present study showed that the three populations of horseshoe crabs were considered to have undergone moderate genetic differentiation with a mean F (ST) value of 0.092 .The current results suggest that allozyme polymorphism in horseshoe crabs is a potential biomonitoring tool for metal contamination, although further validation is required.

Arginine kinase: joint crystallographic and NMR RDC analyses link substrate-associated motions to intrinsic flexibility.

The phosphagen kinase family, including creatine and arginine kinases (AKs), catalyzes the reversible transfer of a "high-energy" phosphate between ATP and a phosphoguanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of AK structures were interpreted as a plastic deformation. Here, the structure of Limulus substrate-free AK is refined against high-resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa) and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.7 Å transition-state analog complex shows large substrate-induced domain motions that can be broken down into movements of smaller quasi-rigid bodies. The solution-state structure of substrate-free AK is most consistent with an equilibrium of substrate-free and substrate-bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, "substrate-induced" motions are along modes that are intrinsically flexible in the substrate-free enzyme and likely involve some degree of conformational selection.

Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin.

Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC(50) of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases.

News from an ancient world: two novel astacin metalloproteases from the horseshoe crab.

In this work, we report the cloning, heterologous expression, and characterization of two novel astacin proteases from the chelicerate Limulus polyphemus (horseshoe crab), designated as LAST (Limulus astacin) and LAST_MAM (Limulus astacin containing a MAM domain), respectively. The expression pattern showed ubiquitous occurrence of LAST_MAM, while LAST was predominantly restricted to the eyes and brain, indicating a function in the nervous system. Both enzymes contain the characteristic metzincin-type zinc-binding region and Met turn. While LAST is made up only of the typical prodomain and astacin-like protease domain, LAST_MAM contains an additional MAM (meprin A5 protein tyrosine phosphatase micro) domain, which so far only has been found in few astacins such as the vertebrate meprin Hydra and squid enzymes, and in a number of other extracellular proteins such as A5 protein and tyrosine phosphatase micro. These gave rise to the designation MAM for this protein module. MAM domains have been shown to be responsible for protein oligomerization in meprin proteases and tyrosine phosphatase micro. Since the horseshoe crab has kept its body plan for almost half a billion years, it is therefore a privileged organism for the study of protease evolution. In this context, we could show by phylogenetic analysis that this protease is not related to the other MAM-domain-containing astacins indicating different evolutionary origins of these proteins. Moreover, we clearly demonstrated the divergent evolvement of the MAM module itself, and not only with regard to proteases. However, there are some unique functional features that are not shared by other members of this protein family. For example, LAST_MAM is the only astacin protease known so far that is active in its zymogen form, indicating that the presence of the N-terminal propeptide does not prevent proteolytic activity.