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A two disulfide bridge Kazal domain from Phytophthora exhibits stable inhibitory activity against serine proteases of the subtilisin family

A two disulfide bridge Kazal domain from Phytophthora exhibits stable inhibitory activity against... Background: Kazal-like serine protease inhibitors are defined by a conserved sequence motif. A typical Kazal domain contains six cysteine residues leading to three disulfide bonds with a 1–5/2– 4/3–6 pattern. Most Kazal domains described so far belong to this class. However, a novel class of Kazal domains with two disulfide bridges resulting from the absence of the third and sixth cysteines have been found in biologically important molecules, such as human LEKTI, a 15-domain inhibitor associated with the severe congenital disease Netherton syndrome. These domains are referred to as atypical Kazal domains. Previously, EPI1, a Kazal-like protease inhibitor from the oomycete plant pathogen Phytophthora infestans, was shown to be a tight-binding inhibitor of subtilisin A. EPI1 also inhibits and interacts with the pathogenesis-related P69B subtilase of the host plant tomato, suggesting a role in virulence. EPI1 is composed of two Kazal domains, the four-cysteine atypical domain EPI1a and the typical domain EPI1b. Results: In this study, we predicted the inhibition constants of EPI1a and EPI1b to subtilisin A using the additivity-based sequence to reactivity algorithm (Laskowski algorithm). The atypical domain EPI1a, but not the typical domain EPI1b, was predicted to have strong inhibitory activity against subtilisin A. Inhibition assays and coimmunoprecipitation experiments showed that recombinant domain EPI1a exhibited stable inhibitory activity against subilisin A and was solely responsible for inhibition and interaction with tomato P69B subtilase. Conclusion: The finding that the two disulfide bridge atypical Kazal domain EPI1a is a stable inhibitor indicates that the missing two cysteines and their corresponding disulfide bond are not essential for inhibitor reactivity and stability. This report also suggests that the Laskowski algorithm originally developed and validated with typical Kazal domains might operate accurately for atypical Kazal domains. specific processes [1]. To regulate the activity of proteases Background Proteases play essential roles in biological systems, not and avoid cellular damage, organisms also produce pro- only digestion and protein turnover but also a diversity of tease inhibitors [1]. So far, 48 distinct families of protease Page 1 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 inhibitors have been described, one of which is the Kazal between Kazal domains and their cognate serine proteases family of serine protease inhibitors (I1 family) [1]. Kazal [12,14-16]. Changes in noncontact residues often do not type inhibitors are widely distributed in animals, apicom- affect equilibrium constants (Ka, the reciprocal of Ki), plexans and oomycetes. They are thought to play impor- whereas changes in contact residues result in significant tant roles in maintenance of normal cellular and alterations of Ka [12]. Among the 12 contact residues, P3, physiological processes of animals [2,3], and pathogene- the second conserved cysteine residue, and P15', a con- sis of mammalian parasitic apicomplexans and plant served asparagine, show little variation in naturally occur- pathogenic oomycetes [4-7]. Kazal-like serine protease ring Kazal domains, but the remaining ten contact inhibitors are defined by a conserved motif in their amino residues are hypervariable [12]. Therefore, the Laskowski acid sequences. Typical Kazal domains contain six algorithm was established based on the residues at the 10 cysteine residues forming a 1–5/2–4/3–6 disulfide bond contact positions and allows for the calculation of Ka or pattern [3,8]. Most Kazal domains described so far belong Ki of a Kazal domain against a selected set of six serine to this class. However, a novel class of Kazal domains has proteases based on the domain sequence alone been described in recent years, in which the third and [12,13,17]. This algorithm was developed based on 191 sixth cysteines are missing resulting in the loss of the 3–6 variants of turkey ovomucoid third domain (19 amino disulfide bond [3,7,9]. These two disulfide bridge acid mutants in the ten contact residues plus the wild domains are referred to here as atypical Kazal domains. type) and was validated with a number of typical Kazal domains [12,13,17]. Theoretically, the algorithm should Atypical Kazal domains were first reported in the human be applicable to atypical Kazal domains since the missing serine proteinase inhibitor LEKTI, a 15-domain inhibitor cysteine residues are different from the hypervariable con- associated with the severe congenital disease Netherton tact residues. However, the accuracy of the algorithm in syndrome [2,9]. Domain 2 and 15 of LEKTI are typical predicting the reactivity of atypical Kazal domains has not Kazal domains with complete 6 cysteine residues, whereas been tested (M. Laskowski, Jr., pers. comm.). the remaining 13 domains represent atypical two disulfide bridge Kazal domains [3,9,10]. The functionality Kazal-like inhibitors are ubiquitous in oomycetes [7], a of some atypical Kazal domains from LEKTI has been group of eukaryotic microbes that includes many devas- examined. Domain 1 of LEKTI does not inhibit any of the tating plant pathogens [18]. A total of 35 putative extracel- standard proteases [11]. Domain 6 exhibits significant lular proteins with 56 predicted Kazal-like domains were inhibitory activity on trypsin, but this inhibition is only identified from five plant pathogenic oomycete species temporary [3,9,11]. A recombinant protein containing [7]. Among them, the Phytophthora infestans Kazal inhibi- four atypical domains of LEKTI (domain 6, 7, 8 and 9) tors EPI1 and EPI10 inhibit and interact with the patho- inhibits both trypsin and subtilisin A permanently [10], genesis-related P69B subtilisin-like serine protease of the indicating that atypical Kazal domains can be effective host plant tomato, suggesting a role in virulence [7,19]. inhibitors. However, it is unclear whether a single atypical Both EPI1 and EPI10 contain an atypical Kazal domain domain can be a stable inhibitor. Multi-domain interac- [7,19]. Atypical domains are common in Kazal-like inhib- tions could be responsible for the stable inhibitory activ- itors of plant pathogenic oomycetes. One-fourth (14/56) ity observed for the recombinant protein [10]. Additional of oomycete Kazal domains belong to this type [7]. These structural and functional studies on atypical Kazal domains are distributed in 14 different proteins from domains are needed to understand the impact of the three Phytophthora species, P. infestans, P. ramorum and P. disulfide bridges on inhibitor activity and stability. sojae, some of which have multiple domains. Remarkably, phylogenetic analysis of the 56 domains revealed that all As a result of exhaustive biochemical studies of the third the atypical domains form a significantly distinct cluster domain of turkey ovomucoid protein performed by the (M. Tian, Z. Liu and S. Kamoun, manuscript in prepara- late Michael Laskowski Jr. and collaborators, much is tion), suggesting that the loss of one disulfide bridge in known about the relationship between domain sequence Phytophthora Kazal-like domains predates speciation. and inhibition specificity in Kazal inhibitor-serine pro- Characterizing the atypical Kazal domains would help to tease interactions. This work culminated in the develop- understand the biochemical and biological functions of ment of an additivity-based sequence to reactivity these inhibitors. algorithm, referred to from here on as the Laskowski algo- rithm, that predicts the inhibition constants (Ki) between P. infestans EPI1 is an ideal candidate to characterize atyp- Kazal domains and a set of six serine proteases based ical Kazal domains. EPI1 was identified as a tight-binding solely on the sequence of the inhibitors [12,13]. Structural inhibitor of subtilisin A and inhibits and interacts with studies of Kazal domain-protease complexes revealed that P69B subtilisin-like serine protease [7]. EPI1 is composed there are 12 contact positions (P6, P5, P4, P3, P2, P1, P1', of two putative Kazal domains, atypical domain EPI1a P2', P3', P14', P15' and P18') responsible for interactions and typical domain EPI1b [7]. The predicted 12 contact Page 2 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 Primar Figure 1 y structure alignment of two Kazal domains of EPI1 and the predicted inhibition constants against subtilisin A Primary structure alignment of two Kazal domains of EPI1 and the predicted inhibition constants against sub- tilisin A. The conserved cysteine residues in both domains are shown in bold. The putative P1-P1' sites and the disulfide link- ages predicted based on the structure of other Kazal domains are shown. The putative 10 hypervariable contact residues are marked with asterisks. The numbers represent the inhibition constants of two Kazal domains against subtilisin A predicted with the Laskowski algorithm. Expression and purification of the two Kazal domains of residues of both domains follow the Kazal consensus, with P3 and P15' conserved cysteines and asparagines, EPI1 respectively, and the remaining 10 contact residues varia- To test the protease inhibitory activities of EPI1a and ble relative to other Kazal domains. In this study, we pre- EPI1b, the two Kazal domains of EPI1, and assess the pre- dicted the inhibition constants of EPI1a and EPI1b to dictions of the Laskowski algorithm, we expressed and subtilisin A using the Laskowski algorithm [12]. The atyp- purified the two recombinant domains in Escherichia coli ical domain EPI1a, but not the typical domain EPI1b, was as fusion proteins with the FLAG epitope tag at the amino- predicted to be a strong inhibitor of subtilisin A. A recom- terminus. The sequences of the recombinant proteins are binant EPI1a exhibited stable inhibitory activity against shown in Fig. 2A. The predicted molecular mass for FLAG- subilisin A and appeared solely responsible for inhibition EPI1a (rEPI1a) and FLAG-EPI1b (rEPI1b) was 10181 Da and interaction with tomato P69B subtilase, providing and 8996 Da, respectively. To determine the purity of the evidence that the missing two cysteine residues and the purified recombinant proteins, we ran 0.5 µg of purified corresponding disulfide bond might not be essential for rEPI1a and rEPI1b on SDS-PAGE gel and stained with sil- inhibitor reactivity and stability. This report also suggests ver nitrate. Bands of the expected sizes were observed for that the additivity-based sequence to reactivity algorithm both proteins. There was only a single band for rEPI1b, (Laskowski algorithm) originally developed and validated indicating high purity (Fig. 2B). The rEPI1a sample with typical Kazal domains might operate accurately for revealed two closely-migrating bands (Fig. 2B). The two atypical domains. bands reacted to the FLAG antibody and are likely to rep- resent rEPI1a with and without the signal peptide OMPA, Results which is located immediately before the FLAG peptide in The atypical Kazal domain of EPI1 is predicted to be a the vector pFLAG-ATS and is responsible for secreted functional inhibitor of subtilisin A expression in E. coli. Similar release of the mature protein The inhibition constants of two Kazal domains of EPI1 was commonly observed with other proteins expressed against subtilisin A were predicted using the Laskowski using pFLAG-ATS (M. Tian and S. Kamoun, unpublished). algorithm [12] based on the sequence of their 10 hyper- We also stained the gel loaded with purified rEPI1a pro- variable contact residues (Fig. 1). Interestingly, the atypi- tein with Coomassie blue. Compared with the band cor- cal Kazal domain EPI1a was predicted to be a strong responding to the rEPI1a without OMPA, the slower- inhibitor of subtilisin A with a Ki of 4.3 nM, a value that migrating band was much weaker (data not shown), indi- is remarkably similar to the experimentally determined Ki cating the secreted version of rEPI1a was the major com- of 2.77 +/- 1.07 nM for the entire EPI1 protein against ponent of the purified rEPI1a protein solution. Besides subtilisin A [7]. In contrast, the typical Kazal domain these two bands, no other proteins were detected by silver EPI1b, which contains the complete set of six cysteine res- staining suggesting that the rEPI1a preparation was highly idues, may not be functional against subtilisin A since the pure. predicted Ki was high at 50 mM. Therefore, these compu- tational analyses predicted that the atypical domain EPI1a is solely responsible for the inhibition of subtilisin A. Page 3 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 H Figure 2 eterologous expression of two Kazal domains of EPI1 Heterologous expression of two Kazal domains of EPI1. A, Amino acid sequences of recombinant Kazal domains rEPI1a and rEPI1b. The letters in upper case represent the amino acid sequence of the EPI1 protein. Residues in bold correspond to the native Kazal domains EPI1a and EPI1b as shown in Fig. 1. The letters in lower case represent the vector derived sequence with the underlined ones representing FLAG epitope tag. Numbers indicate the position of amino acid residues starting from the N terminus of EPI1 protein. B, Affinity purified recombinant Kazal domains visualized on SDS-PAGE stained with silver nitrate. The rEPI1a with the signal peptide OMPA is indicated by an asterisk. The numbers on the left represent the size of molecular weight markers. The atypical Kazal domain EPI1a inhibits the serine P69B, or A. tumefaciens containing the empty binary vec- protease subtilisin A tor pCB302-3. 10 µl of intercellular fluids from the two We performed inhibition assays of subtilisin A by incubat- treatments were used in in-gel protease assays. A distinct ing 0.2 µM of subtilisin A with 0.2 µM of rEPI1a, rEPI1b additional protease band was observed in the P69B- or buffer control in a volume of 50 µl. The remaining pro- expressing sample but not in the control suggesting that tease activity was measured using the QuantiCleave™ Pro- P69B-HA is functional (Fig. 4A). 10 µl of P69B-expressing tease Assay Kit as described in methods. In repeated N. benthamiana intercellular fluids were incubated with 20 assays, rEPI1a was found to inhibit about 91% of the pmol of the EPI1 recombinant protein rEPI1 [7], rEPI1a, measured activity of subtilisin A, whereas rEPI1b did not rEPI1b, or buffer and the remaining protease activity was display any significant inhibition (Fig. 3). These results detected by in-gel protease assay. rEPI1a, containing the are consistent with the prediction of the Laskowski algo- atypical Kazal domain, completely inhibited the P69B rithm (Fig. 1). It is unlikely that the FLAG tag interfered band similar to rEPI1. rEPI1 and rEPI1a also inhibited the with the inhibitory activities considering that both the activity of two other extracellular proteases from N. active rEPI1a and inactive rEPI1b carry the FLAG sequence benthamiana (Fig. 4B). The identity of these N. benthami- and that these experiments were conducted in parallel. ana proteases is unknown but they could also be subtili- sin-like serine proteases, such as homologs of tomato P69. EPI1a inhibits the tomato pathogenesis-related P69B In these experiments, rEPI1b did not exhibit any inhibi- subtilase tion towards P69B or other protease bands. EPI1 inhibits and interacts with the pathogenesis-related P69B subtilase of tomato [7]. To test which of the two EPI1a interacts with tomato P69 domains of EPI1 inhibits P69B, we first used agroinfiltra- We previously showed that rEPI1 interacts with P69B [7]. tion to transiently express P69B fused with the epitope tag Here, we tested whether the atypical Kazal domain EPI1a HA at the C-terminus in Nicotiana benthamiana leaves. interacts with P69B subtilase by coimmunoprecipitation. Intercellular fluids were collected from leaves infiltrated Coimmunoprecipitation was performed on BTH-induced with either Agrobacterium tumefaciens containing pCB302- tomato intercellular fluids incubated with rEPI1a, rEPI1b Page 4 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 The Figure 3 atypical Kazal domain EPI1a inhibits subtilisin A The atypical Kazal domain EPI1a inhibits subtilisin A. The remaining protease activity of subtilisin A was measured The atypical Ka Figure 4 zal domain rEPI1a inhibits P69B subtilase after incubating with rEPI1a, rEPI1b or without protease The atypical Kazal domain rEPI1a inhibits P69B sub- inhibitors (Std) using the QuantiCleave™ Protease Assay Kit tilase. A, In-gel protease assay of Nicotiana benthamiana as described in the methods. Activity is expressed as a per- intercellular fluids expressing the empty binary vector centage of total protease activity in the absence of protease pCB302-3 (-) or pCB-P69B (+). B, Inhibition assay of P69B by inhibitors. The bars correspond to the mean of three inde- recombinant EPI1 entire protein and the single Kazal pendent experiments with three replications for each experi- domains. P69B-expressing N. benthamiana intercellular fluids ment. The error bars represent the standard errors were incubated in the presence of rEPI1, rEPI1a, rEPI1b or calculated from the mean of three experiments. the absence of protease inhibitors (Buffer) and then the remaining protease activity was analyzed using zymogen in- gel protease assays. The arrow indicates the band location corresponding to the protease activity of P69B. or buffer control using FLAG antibody covalently linked agarose beads. Western blots were hybridized sequentially with P69 and FLAG antisera and revealed that P69 subti- lases co-precipitated with rEPI1a (Fig. 5). This indicates assays with varying concentrations of EPI1a (Fig. 6A). The that rEPI1a interacts with P69 subtilases. Since the P69 concentration of 0.15 µM of EPI1a resulted in inhibition family of subtilases have at least six homologs (P69A- levels of about 80% of the measured protease activity and P69F) [20,21] and the peptide used to generate the P69 was selected for the stability analysis. This concentration antisera is conserved among several homologs [7], we is within the linear part of the curve (Fig. 6A), suggesting cannot conclude that P69B subtilase is the only protein that hydrolysis of rEPI1a could be easily detected as a that was pulled down with rEPI1a. In contrast to rEPI1a, decrease in inhibitory activity. The inhibitory activity of rEPI1b could not be detected after coimmunoprecipita- rEPI1a did not show any decrease over 3 hours of incuba- tion with BTH-induced tomato intercellular fluids (Fig. tion with subtilisin A (Fig. 6B), indicating that EPI1a is a 5). However, rEPI1b was detected in control coimmuno- stable inhibitor of subtilisin A. These results are in sharp precipitations with the extraction buffer of tomato inter- contrast with those reported for domain LD-6 of LEKTI, cellular fluids (data not shown), suggesting that this which lost 50% of inhibitory activity after 1 hour of incu- protein is not stable in BTH-induced tomato intercellular bation with trypsin and lost all inhibitory effect after 3–4 fluids. hours [3]. EPI1a exhibits stable inhibitory activity Discussion To determine whether EPI1a is a temporary or stable Atypical Kazal domains with two disulfide bridges occur inhibitor of subtilisin, we performed stability analyses by in biologically important molecules. The 15-domain incubating subtilisin A with or without rEPI1a for increas- human serine protease inhibitor LEKTI that carries 13 ing periods of time and measuring the remaining protease atypical Kazal domains is associated with the severe con- activity. To determine the optimal concentration of EPI1a genital disorder Netherton syndrome [2]. The protease for the stability analyses, we first performed inhibition inhibitors EPI1 and EPI10 of P. infestans contain an Page 5 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 Coim a Figure 5 nd P6 munop 9 subtrilases using FLAG a ecipitation of the recomb ntiserainant Kazal domains Coimmunoprecipitation of the recombinant Kazal domains and P69 subtilases using FLAG antisera. Elu- ates from coimmunoprecipitation of rEPI1a, rEPI1b or buffter with proteins in BTH-treated tomato intercellular fluids were run on SDS-PAGE gel followed by sequential immunobloting with P69 (α-P69) and FLAG (α-FLAG) antisera at a dilution of 1:3000. atypical Kazal domain and have been implicated in viru- The atypical Kaz activity Figure 6 against subtilisin A al domain rEPI1a exhibits stable inhibitory lence of this devastating plant pathogen [7,19]. Although The atypical Kazal domain rEPI1a exhibits stable the structure and function of atypical Kazal domains from inhibitory activity against subtilisin A. A, Protease activ- LEKTI have been studied, the effects of the loss of Cys 3, ity of subtilisin A (0.2 µM) in the presence of rEPI1a in con- Cys 6 and the corresponding disulfide bond on inhibitor centrations ranging from 2 nM to 0.3 µM. Activity is expressed as a percentage of total protease activity in the reactivity and stability was not assessed and there is no absence of protease inhibitors. B, Protease activity of subtili- evidence showing that a single atypical Kazal domain can sin A (0.2 µM) after preincubation with 0.15 µM of rEPI1a be a stable inhibitor by itself [3,10,11]. In this study, we (black column) or without protease inhibitors (gray column) describe that the atypical domain EPI1a of the two- for a period of time ranging from 30 min to 180 min. Activity domain EPI1 protein is a stable inhibitor of the subtilisin is expressed as a percentage of total protease activity in the family of serine proteases. No loss of inhibitory activity absence of protease inhibitors at each treatment. The bars was found even after incubating EPI1a with subtilisin A correspond to the mean of three independent replications of for 3 hours. The loss of Cys 3, Cys 6 and the corresponding one representative experiment out of three performed. The disulfide bond does not have major adverse effects on error bars represent the standard errors calculated from the inhibitory activity or stability, indicating that these two three replications. cysteine residues might not be essential for the function of Kazal domains. This finding is important for determining the biochemical and biological functions of Kazal inhibi- tors containing atypical Kazal domain(s). Kazal-like pro- teins have been reported from animals, apicomplexans, atypical Kazal domains have been identified in plant oomycetes, as well as the bacterium Nitrosomonas europaea pathogenic oomycetes [7]. [7]. The availability of genomic sequence from a diversity of organisms is likely to reveal an increasing number of The structural mechanism underlying the stability of atypical Kazal inhibitors. For example, so far, a total of 14 EPI1a is not clear. The three-dimensional structure of the Page 6 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 atypical domain 6 (LD6) of LEKTI was determined. The total of 56 Kazal-like domains identified in five plant overall structure of LD6 resembles the three-dimensional pathogenic oomycetes have only four cysteines. Two of fold of typical Kazal-type inhibitors, but the backbone these Kazal-like inhibitors EPI1 and EPI10 of P. infestans geometry of its canonical loop is not well defined, provid- target the defense-related protease P69B of the host plant ing a possible explanation for its temporary inhibitory tomato. The first atypical Kazal domain of EPI1 appears to activity [11]. There are 13 residues between the first be solely functional in inhibiting and interacting with cysteine residue (Cys 1) and the second one (Cys 2) in P69B. In the three-domain EPI10, the second domain is LD6, instead of 6–9 in most typical Kazal domains [11]. also an atypical domain that was predicted to be func- The lack of one disulfide bond and the longer sequence tional against subtilisin based on the Laskowski algorithm stretch between the first two cysteines were proposed to be [19]. These findings raise some interesting questions. the factors responsible for the instability of LD6 [11]. What are the biochemical and biological implications of Indeed, the longer sequence stretch between the first two the loss of the disulfide bridge? Are there any evolutionary cysteines could explain the difference between EPI1a and advantages of the two-disulfide bridge Kazal domain over LD6. There are only 3 residues between Cys 1 and Cys 2 in the three-disulfide bridge domain in counteracting and EPI1a, which is shorter than in most Kazal domains. The co-evolving with host proteases? Additional functional longer sequence stretch of LD6 might lead to the abnor- and structural studies are needed to address these mal canonical loop and the non-permanent inhibitory questions. activity. Future work to determine the three-dimensional structure of EPI1a and compare it with LD6 of LEKTI and Conclusion other Kazal domains should help to unravel the structural In this study, the functionality of a two disulfide bridge mechanism underlying the functionality of atypical Kazal atypical Kazal domain EPI1a from Phytophthora was char- domains. acterized. EPI1a was predicted to be a strong inhibitor of subtilisin A using the additivity-based sequence to reactiv- The Laskowski algorithm was developed and validated ity algorithm (Laskowski algorithm). Inhibition assays based on typical Kazal domains [12,13]. The algorithm and coimmunoprecipitation experiments showed that exploits an exhaustive analysis of all amino acid variants recombinant domain EPI1a exhibited stable inhibitory in the ten hypervariable contact residues of turkey ovomu- activity against subilisin A and was solely responsible for coid third domain, a typical Kazal domain. The accuracy inhibition and interaction with tomato P69B subtilase, of the algorithm in predicting the reactivity of atypical providing evidence that the missing two cysteines and Kazal domains has not been evaluated (M. Laskowski Jr., their corresponding disulfide bond are not essential for pers. comm.). Here we found that the algorithm correctly inhibitor reactivity and stability. This report also suggests predicted which of the two EPI domains is likely to inhibit that the Laskowski algorithm originally developed and subtilisins. Our experimental data showed that the atypi- validated with typical Kazal domains might operate accu- cal Kazal domain EPI1a inhibited subtilisin A, and inhib- rately for atypical Kazal domains. ited and interacted with P69 subtilase similar to the entire EPI1 protein [7]. Also, using the algorithm, the atypical Methods Kazal domain EPI1a was predicted to be a strong inhibitor Prediction of inhibition constants of subtilisin A with a predicted Ki of 4.3 nM, which was in The putative ten hypervariable contact residues of EPI1a and EPI1b were identified based on similarity to canoni- very good agreement with the experimentally determined Ki of 2.77 +/- 1.07nM [7]. As expected from the predicted cal animal Kazal domains [14-16] and are shown in Fig. Ki of 50 mM, the typical EPI1b domain was not an effec- 1. Predicted inhibition constants for the EPI1 domains tive inhibitor of subtilisin A. In summary, it appears that against subtilisin A (Carlsberg) were generated by Drs. M. the Laskowski algorithm operates accurately for atypical A. Qasim and M. Laskowski Jr., Purdue University, with Kazal domains such as EPI1a. Perhaps, this is expected the additivity-based sequence to reactivity algorithm since the Cys 3 and Cys 6 residues of typical Kazal (Laskowski algorithm) described by Lu et al. [12]. domains are not contact positions. Nonetheless, our Plant growth and BTH treatment observations and the concordance between predicted and experimental data suggest that gross structural changes Tomato (Lycopersicon esculentum) cultivar Ohio 7814 and that could result from the loss of one disulfide bridge in N. benthamiana plants were grown in pots at 25°C, 60% atypical Kazal domains may not affect the specificity of humidity, under 16 hour-light/8 hour-dark cycle. We used the interactions between Kazal domains and their cognate the salicylic acid analog benzo-(1,2,3)-thiadiazole-7-car- serine proteases. bothioic acid S-methyl ester (BTH) to induce PR proteins. BTH treatment of tomato plants followed the exact same Atypical Kazal domains are ubiquitous in serine protease procedure described previously [7]. inhibitors of plant pathogenic oomycetes. Fourteen of a Page 7 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 Bacterial strains and plasmids ously [26]. A. tumefaciens strains carrying plasmids pCB- E. coli XL1-Blue and A. tumefaciens GV3101 were used in P69B, empty vector pCB302-3 [23], and pCB301-P19 (J. this study and were routinely grown in Luria-Bertani (LB) Win and S. Kamoun, unpublished) were used. pCB301- media [22] at 37°C and 28°C, respectively. Plasmids P19 is a construct expressing the P19 protein of tomato pFLAG-EPI1a and pFLAG-EPI1b for protein expression bushy stunt virus (TBSV), a suppressor of post-transcrip- were constructed by cloning the PCR amplified DNA frag- tional gene silencing in N. benthamiana that significantly ments corresponding to the coding sequence of Kazal enhances in planta transient expression [27]. Overnight domains EPI1a and EPI1b together with some flanking agrobacteria cultures were harvested by centrifugation at sequence into EcoRI and KpnI sites of pFLAG-ATS (Sigma, 2000 g for 20 min, and resuspended in 10 mM MgCl , 10 St. Louis, MO), a vector that allows secreted expression in mM MES (pH 5.6) and 150 µM acetosyringone. Resus- E. coli. The primers used for amplification of epi1a are pended agrobacteria cultures of pCB-P69B or pCB302-3 epi1a-F1(5'-gcggaattcTCAAAGCCCGCAAGTCATCAG-3') with an optical density (OD ) of 1.0 were mixed with and epi1a-R1(5'-gcgggtaccTTACTTGCTGGGAGGCT- equal volumes of a culture of pCB301-P19 with an optical GCTCGCCAG-3'). The primers used for amplification of density (OD ) of 2.0. The mixtures were kept at room CACCGGTAGCTCCACT- epi1b are epi1b-F1(5'-gcggaattc temperature for 3 hours and then infiltrated into leaves of GGCGAGCAGC-3') and epi1b-R1(5'-gcgggtaccTTATC- 6-week-old N. benthamiana plants. Intercellular fluids CCTCCTGCGGTGTC-3'). The introduced EcoRI and KpnI from infiltrated leaves were isolated 5 days after restriction sites for cloning are underlined. The letters in infiltration. upper case represent gene specific sequence. The detailed sequence information for the expressed fusion proteins Isolation of intercellular fluids FLAG-EPI1a (rEPI1a) and FLAG-EPI1b (rEPI1b) is shown Intercellular fluids were prepared from tomato and N. in Fig. 2A. Plasmid pCB-P69B is a construct with the open bethamiana leaves according to the method of de Wit and reading frame of the P69B gene [GenBank: Y17276] fused Spikman [28]. For tomato leaves, a 0.24 M sorbitol solu- with the HA tag (YPYDVPDYA) at the C-terminus cloned tion was used as extraction buffer. For leaves from N. into the binary vector pCB302-3 [23] and was described benthamiana, a solution of 300 mM NaCl, 50 mM NaPO elsewhere [19]. pH 7 [26] was used as extraction buffer. The intercellular fluids were filter sterilized (0.45 µM), and were used SDS-PAGE and Western blot analyses immediately or stored at -20°C. Proteins were subjected to 15% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) as previ- In-gel protease assays ously described [22]. Following electrophoresis, gels were In-gel protease assays were performed with 10% SDS- stained with silver nitrate following the method of Merril polyacrylamide gel containing 0.1% (w/v) gelatin (Bio- et al. [24] or with Coomassie Brilliant Blue [22], or the Rad Laboratories, Hercules, CA) using BIO-RAD's zymo- proteins were transferred to supported nitrocellulose gram buffer system as described earlier [7]. membranes (BioRad Laboratories, Hercules, CA) using a Mini Trans-Blot apparatus (BioRad Laboratories, Her- Inhibition assays of subtilisin A by EPI1 Kazal domains cules, CA). Detection of antigen-antibody complexes was Inhibition assays of subtilisin A by EPI1 Kazal domains carried out with a Western blot alkaline phosphatase kit were performed using colorimetric QuantiCleave™ Pro- (BioRad Laboratories, Hercules, CA). Antisera to P69 sub- tease Assay Kit (Pierce, Rockford, IL). 0.2 µM of subtilisin tilases were raised against a peptide specific for the tomato A (Carlsberg) (Sigma, St. Luis, MO) was preincubated P69 family [7]. Monoclonal anti-FLAG M2 antibody was with different amount of purified EPI1 Kazal domains, in purchased from Sigma (St. Louis, MO). a volume of 50 µl buffer for 30 min at 25°C, and then the remaining protease activity was measured following the Expression and purification of rEPI1a and rEPI1b procedures as described previously [7]. Analysis of the sta- Expression and purification of rEPI1a and rEPI1b was ble inhibitory activity of rEPI1a against subtilisin A was conducted as described previously for other pFLAG-ATS performed by incubating 0.2 µM of subtilisin A with 0.15 derived constructs [7,25]. Protein concentrations were µM of rEPI1a in 50 µl buffer (50 mM Tris, pH 8.0) for a determined using the BioRad protein assay (BioRad Labo- time period of 0–180 min at 25°C and then measuring ratories, Hercules, CA). To determine the purity, 0.5 µg of residue enzyme activity. the purified protein was run on a SDS-PAGE gel followed Coimmunoprecipitation by staining with silver nitrate. Coimmunoprecipitation of Kazal domains rEPI1a and Transient expression of P69B subtilase in planta rEPI1b with BTH-treated tomato intercellular fluids was Transient expression of P69B-HA in planta was performed performed using the FLAG-tagged protein immunoprecip- according to the agroinfiltration method described previ- itation kit (Sigma, St. Luis, MO) as described previously Page 8 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 T, Lin TY, Ogawa M, Otlewski J, Park SJ, Qasim S, Ranjbar M, Tashiro [7]. 100 pmol of purified rEPI1a or rEPI1b were preincu- M, Warne N, Whatley H, Wieczorek A, Wieczorek M, Wilusz T, bated with 300 µl of tomato intercellular fluids for 30 min Wynn R, Zhang W, Laskowski MJ: Predicting the reactivity of at 25°C. 40 µl of anti-FLAG M2 resin was added and incu- proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases. Proc Natl Acad Sci U S A 2001, bated at 4°C for 2 h with gentle shaking. The precipitated 98:1410-1415. protein complexes were eluted in 60 µl of FLAG peptide 13. Laskowski MJ, Qasim MA, Yi Z: Additivity-based prediction of equilibrium constants for some protein-protein associations. solution (150 ng/µl) and were analyzed by SDS-PAGE Curr Opin Struct Biol 2003, 13:130-139. and Western blot analyses. 14. Read RJ, Fujinaga M, Sielecki AR, James MN: Structure of the com- plex of Streptomyces griseus protease B and the third domain of the turkey ovomucoid inhibitor at 1.8-A Authors' contributions resolution. Biochemistry 1983, 22:4420-4433. MT, designing and performance of wet lab experiments, 15. Lu W, Apostol I, Qasim MA, Warne N, Wynn R, Zhang WL, Ander- writing of manuscript. SK, supervision of experimental son S, Chiang YW, Ogin E, Rothberg I, Ryan K, Laskowski MJ: Bind- ing of amino acid side-chains to S1 cavities of serine work, writing of manuscript. proteinases. J Mol Biol 1997, 266:441-461. 16. Laskowski MJ, Kato I, Ardelt W, Cook J, Denton A, Empie MW, Kohr WJ, Park SJ, Parks K, Schatzley BL, et al.: Ovomucoid third Acknowledgements domains from 100 avian species: isolation, sequences, and We are grateful to Dr. Michael Laskowski Jr. and Dr. M. A. Qasim from Pur- hypervariability of enzyme-inhibitor contact residues. Bio- due University for predicting the inhibition constants and sharing their chemistry 1987, 26:202-221. expert knowledge on Kazal inhibitors. We also thank Diane Kinney for 17. Qasim MA, Lu W, Lu SM, Ranjbar M, Yi Z, Chiang YW, Ryan K, Anderson S, Zhang W, Qasim S, Laskowski MJ: Testing of the addi- technical assistance and three anonymous reviewers for useful suggestions. tivity-based protein sequence to reactivity algorithm. Bio- This work was supported by USDA-NRI project OHO00963-SS. Salaries chemistry 2003, 42:6460-6466. and research support were provided by State and Federal Funds appropri- 18. Kamoun S: Molecular genetics of pathogenic oomycetes. ated to the Ohio Agricultural Research and Development Center, the Ohio Eukaryotic Cell 2003, 2:191-199. State University. 19. Tian M, Benedetti B, Kamoun S: A second Kazal-like protease inhibitor from Phytophthora infestans inhibits and interacts with the apoplastic pathogenesis-related protease P69B of References tomato. Plant Physiology 2005, 138:1785-1793. 1. Rawlings ND, Tolle DP, Barrett AJ: Evolutionary families of 20. Jorda L, Coego A, Conejero V, Vera P: A genomic cluster contain- peptidase inhibitors. Biochem J 2004, 378:705-716. ing four differentially regulated subtilisin-like processing pro- 2. Magert HJ, Kreutzmann P, Standker L, Walden M, Drogemuller K, tease genes is in tomato plants. J Biol Chem 1999, Forssmann WG: LEKTI: a multidomain serine proteinase 274:2360-2365. inhibitor with pathophysiological relevance. Int J Biochem Cell 21. Jorda L, Conejero V, Vera P: Characterization of P69E and P69F, Biol 2002, 34:573-576. two differentially regulated genes encoding new members of 3. Kreutzmann P, Schulz A, Standker L, Forssmann WG, Magert HJ: the subtilisin-like proteinase family from tomato plants. Plant Recombinant production, purification and biochemical char- Physiol 2000, 122:67-73. acterization of domain 6 of LEKTI: a temporary Kazal-type- 22. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Labora- related serine proteinase inhibitor. J Chromatogr B Analyt Technol tory Manual. 2nd edition. Cold Spring Harbor, N.Y., Cold Spring Biomed Life Sci 2004, 803:75-81. Harbor Laboratory; 1989. 4. Pszenny V, Ledesma BE, Matrajt M, Duschak VG, Bontempi EJ, 23. Xiang C, Han P, Lutziger I, Wang K, Oliver DJ: A mini binary vec- Dubremetz JF, Angel SO: Subcellular localization and post- tor series for plant transformation. Plant Mol Biol 1999, secretory targeting of TgPI, a serine proteinase inhibitor 40:711-717. from Toxoplasma gondii. Mol Biochem Parasitol 2002, 24. Merril CR, Goldman D, Sedman SA, Ebert MH: Ultrasensitive stain 121:283-286. for proteins in polyacrylamide gels shows regional variation 5. Pszenny V, Angel SO, Duschak VG, Paulino M, Ledesma B, Yabo MI, in cerebrospinal fluid proteins. Science 1981, 211:1437-1438. Guarnera E, Ruiz AM, Bontempi EJ: Molecular cloning, sequenc- 25. Kamoun S, van West P, de Jong AJ, de Groot K, Vleeshouwers V, ing and expression of a serine proteinase inhibitor gene from Govers F: A gene encoding a protein elicitor of Phytophthora Toxoplasma gondii. Mol Biochem Parasitol 2000, 107:241-249. infestans is down-regulated during infection of potato. Mol 6. Morris MT, Cheng WC, Zhou XW, Brydges SD, Carruthers VB: Plant-Microbe Interact 1997, 10:13-20. Neospora caninum expresses an unusual single-domain 26. Kruger J, Thomas CM, Golstein C, Dixon MS, Smoker M, Tang S, Kazal protease inhibitor that is discharged into the parasito- Mulder L, Jones JD: A tomato cysteine protease required for phorous vacuole. Int J Parasitol 2004, 34:693-701. Cf-2-dependent disease resistance and suppression of 7. Tian M, Huitema E, Da Cunha L, Torto-Alalibo T, Kamoun S: A autonecrosis. Science 2002, 296:744-747. Kazal-like extracellular serine protease inhibitor from Phy- 27. Voinnet O, Rivas S, Mestre P, Baulcombe D: An enhanced tran- tophthora infestans targets the tomato pathogenesis- sient expression system in plants based on suppression of related protease P69B. J Biol Chem 2004, 279:26370-26377. gene silencing by the p19 protein of tomato bushy stunt 8. Laskowski MJ, Kato I: Protein inhibitors of proteinases. Annu Rev virus. Plant J 2003, 33:949-956. Biochem 1980, 49:593-626. 28. de Wit PJGM, Spikman G: Evidence for the occurence of race 9. Magert HJ, Standker L, Kreutzmann P, Zucht HD, Reinecke M, Som- and cultivar-specific elicitors of necrosis in intercellular fluids merhoff CP, Fritz H, Forssmann WG: LEKTI, a novel 15-domain of compatible interactions of Cladosporium fulvum and type of human serine proteinase inhibitor. J Biol Chem 1999, tomato. Physiol Plant Pathol 1982, 21:1-11. 274:21499-21502. 10. Jayakumar A, Kang Y, Mitsudo K, Henderson Y, Frederick MJ, Wang M, El-Naggar AK, Marx UC, Briggs K, Clayman GL: Expression of LEKTI domains 6-9' in the baculovirus expression system: recombinant LEKTI domains 6-9' inhibit trypsin and subtili- sin A. Protein Expr Purif 2004, 35:93-101. 11. Lauber T, Schulz A, Schweimer K, Adermann K, Marx UC: Homol- ogous proteins with different folds: the three-dimensional structures of domains 1 and 6 of the multiple Kazal-type inhibitor LEKTI. J Mol Biol 2003, 328:205-219. 12. Lu SM, Lu W, Qasim MA, Anderson S, Apostol I, Ardelt W, Bigler T, Chiang YW, Cook J, James MN, Kato I, Kelly C, Kohr W, Komiyama Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Biochemistry Springer Journals

A two disulfide bridge Kazal domain from Phytophthora exhibits stable inhibitory activity against serine proteases of the subtilisin family

Tian, Miaoying; Kamoun, Sophien
BMC Biochemistry , Volume 6 (1) – Aug 23, 2005
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Springer Journals
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Copyright © 2005 by Tian and Kamoun; licensee BioMed Central Ltd.
Subject
Life Sciences; Biochemistry, general; Life Sciences, general
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1471-2091
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10.1186/1471-2091-6-15
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16117831
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Abstract

Background: Kazal-like serine protease inhibitors are defined by a conserved sequence motif. A typical Kazal domain contains six cysteine residues leading to three disulfide bonds with a 1–5/2– 4/3–6 pattern. Most Kazal domains described so far belong to this class. However, a novel class of Kazal domains with two disulfide bridges resulting from the absence of the third and sixth cysteines have been found in biologically important molecules, such as human LEKTI, a 15-domain inhibitor associated with the severe congenital disease Netherton syndrome. These domains are referred to as atypical Kazal domains. Previously, EPI1, a Kazal-like protease inhibitor from the oomycete plant pathogen Phytophthora infestans, was shown to be a tight-binding inhibitor of subtilisin A. EPI1 also inhibits and interacts with the pathogenesis-related P69B subtilase of the host plant tomato, suggesting a role in virulence. EPI1 is composed of two Kazal domains, the four-cysteine atypical domain EPI1a and the typical domain EPI1b. Results: In this study, we predicted the inhibition constants of EPI1a and EPI1b to subtilisin A using the additivity-based sequence to reactivity algorithm (Laskowski algorithm). The atypical domain EPI1a, but not the typical domain EPI1b, was predicted to have strong inhibitory activity against subtilisin A. Inhibition assays and coimmunoprecipitation experiments showed that recombinant domain EPI1a exhibited stable inhibitory activity against subilisin A and was solely responsible for inhibition and interaction with tomato P69B subtilase. Conclusion: The finding that the two disulfide bridge atypical Kazal domain EPI1a is a stable inhibitor indicates that the missing two cysteines and their corresponding disulfide bond are not essential for inhibitor reactivity and stability. This report also suggests that the Laskowski algorithm originally developed and validated with typical Kazal domains might operate accurately for atypical Kazal domains. specific processes [1]. To regulate the activity of proteases Background Proteases play essential roles in biological systems, not and avoid cellular damage, organisms also produce pro- only digestion and protein turnover but also a diversity of tease inhibitors [1]. So far, 48 distinct families of protease Page 1 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 inhibitors have been described, one of which is the Kazal between Kazal domains and their cognate serine proteases family of serine protease inhibitors (I1 family) [1]. Kazal [12,14-16]. Changes in noncontact residues often do not type inhibitors are widely distributed in animals, apicom- affect equilibrium constants (Ka, the reciprocal of Ki), plexans and oomycetes. They are thought to play impor- whereas changes in contact residues result in significant tant roles in maintenance of normal cellular and alterations of Ka [12]. Among the 12 contact residues, P3, physiological processes of animals [2,3], and pathogene- the second conserved cysteine residue, and P15', a con- sis of mammalian parasitic apicomplexans and plant served asparagine, show little variation in naturally occur- pathogenic oomycetes [4-7]. Kazal-like serine protease ring Kazal domains, but the remaining ten contact inhibitors are defined by a conserved motif in their amino residues are hypervariable [12]. Therefore, the Laskowski acid sequences. Typical Kazal domains contain six algorithm was established based on the residues at the 10 cysteine residues forming a 1–5/2–4/3–6 disulfide bond contact positions and allows for the calculation of Ka or pattern [3,8]. Most Kazal domains described so far belong Ki of a Kazal domain against a selected set of six serine to this class. However, a novel class of Kazal domains has proteases based on the domain sequence alone been described in recent years, in which the third and [12,13,17]. This algorithm was developed based on 191 sixth cysteines are missing resulting in the loss of the 3–6 variants of turkey ovomucoid third domain (19 amino disulfide bond [3,7,9]. These two disulfide bridge acid mutants in the ten contact residues plus the wild domains are referred to here as atypical Kazal domains. type) and was validated with a number of typical Kazal domains [12,13,17]. Theoretically, the algorithm should Atypical Kazal domains were first reported in the human be applicable to atypical Kazal domains since the missing serine proteinase inhibitor LEKTI, a 15-domain inhibitor cysteine residues are different from the hypervariable con- associated with the severe congenital disease Netherton tact residues. However, the accuracy of the algorithm in syndrome [2,9]. Domain 2 and 15 of LEKTI are typical predicting the reactivity of atypical Kazal domains has not Kazal domains with complete 6 cysteine residues, whereas been tested (M. Laskowski, Jr., pers. comm.). the remaining 13 domains represent atypical two disulfide bridge Kazal domains [3,9,10]. The functionality Kazal-like inhibitors are ubiquitous in oomycetes [7], a of some atypical Kazal domains from LEKTI has been group of eukaryotic microbes that includes many devas- examined. Domain 1 of LEKTI does not inhibit any of the tating plant pathogens [18]. A total of 35 putative extracel- standard proteases [11]. Domain 6 exhibits significant lular proteins with 56 predicted Kazal-like domains were inhibitory activity on trypsin, but this inhibition is only identified from five plant pathogenic oomycete species temporary [3,9,11]. A recombinant protein containing [7]. Among them, the Phytophthora infestans Kazal inhibi- four atypical domains of LEKTI (domain 6, 7, 8 and 9) tors EPI1 and EPI10 inhibit and interact with the patho- inhibits both trypsin and subtilisin A permanently [10], genesis-related P69B subtilisin-like serine protease of the indicating that atypical Kazal domains can be effective host plant tomato, suggesting a role in virulence [7,19]. inhibitors. However, it is unclear whether a single atypical Both EPI1 and EPI10 contain an atypical Kazal domain domain can be a stable inhibitor. Multi-domain interac- [7,19]. Atypical domains are common in Kazal-like inhib- tions could be responsible for the stable inhibitory activ- itors of plant pathogenic oomycetes. One-fourth (14/56) ity observed for the recombinant protein [10]. Additional of oomycete Kazal domains belong to this type [7]. These structural and functional studies on atypical Kazal domains are distributed in 14 different proteins from domains are needed to understand the impact of the three Phytophthora species, P. infestans, P. ramorum and P. disulfide bridges on inhibitor activity and stability. sojae, some of which have multiple domains. Remarkably, phylogenetic analysis of the 56 domains revealed that all As a result of exhaustive biochemical studies of the third the atypical domains form a significantly distinct cluster domain of turkey ovomucoid protein performed by the (M. Tian, Z. Liu and S. Kamoun, manuscript in prepara- late Michael Laskowski Jr. and collaborators, much is tion), suggesting that the loss of one disulfide bridge in known about the relationship between domain sequence Phytophthora Kazal-like domains predates speciation. and inhibition specificity in Kazal inhibitor-serine pro- Characterizing the atypical Kazal domains would help to tease interactions. This work culminated in the develop- understand the biochemical and biological functions of ment of an additivity-based sequence to reactivity these inhibitors. algorithm, referred to from here on as the Laskowski algo- rithm, that predicts the inhibition constants (Ki) between P. infestans EPI1 is an ideal candidate to characterize atyp- Kazal domains and a set of six serine proteases based ical Kazal domains. EPI1 was identified as a tight-binding solely on the sequence of the inhibitors [12,13]. Structural inhibitor of subtilisin A and inhibits and interacts with studies of Kazal domain-protease complexes revealed that P69B subtilisin-like serine protease [7]. EPI1 is composed there are 12 contact positions (P6, P5, P4, P3, P2, P1, P1', of two putative Kazal domains, atypical domain EPI1a P2', P3', P14', P15' and P18') responsible for interactions and typical domain EPI1b [7]. The predicted 12 contact Page 2 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 Primar Figure 1 y structure alignment of two Kazal domains of EPI1 and the predicted inhibition constants against subtilisin A Primary structure alignment of two Kazal domains of EPI1 and the predicted inhibition constants against sub- tilisin A. The conserved cysteine residues in both domains are shown in bold. The putative P1-P1' sites and the disulfide link- ages predicted based on the structure of other Kazal domains are shown. The putative 10 hypervariable contact residues are marked with asterisks. The numbers represent the inhibition constants of two Kazal domains against subtilisin A predicted with the Laskowski algorithm. Expression and purification of the two Kazal domains of residues of both domains follow the Kazal consensus, with P3 and P15' conserved cysteines and asparagines, EPI1 respectively, and the remaining 10 contact residues varia- To test the protease inhibitory activities of EPI1a and ble relative to other Kazal domains. In this study, we pre- EPI1b, the two Kazal domains of EPI1, and assess the pre- dicted the inhibition constants of EPI1a and EPI1b to dictions of the Laskowski algorithm, we expressed and subtilisin A using the Laskowski algorithm [12]. The atyp- purified the two recombinant domains in Escherichia coli ical domain EPI1a, but not the typical domain EPI1b, was as fusion proteins with the FLAG epitope tag at the amino- predicted to be a strong inhibitor of subtilisin A. A recom- terminus. The sequences of the recombinant proteins are binant EPI1a exhibited stable inhibitory activity against shown in Fig. 2A. The predicted molecular mass for FLAG- subilisin A and appeared solely responsible for inhibition EPI1a (rEPI1a) and FLAG-EPI1b (rEPI1b) was 10181 Da and interaction with tomato P69B subtilase, providing and 8996 Da, respectively. To determine the purity of the evidence that the missing two cysteine residues and the purified recombinant proteins, we ran 0.5 µg of purified corresponding disulfide bond might not be essential for rEPI1a and rEPI1b on SDS-PAGE gel and stained with sil- inhibitor reactivity and stability. This report also suggests ver nitrate. Bands of the expected sizes were observed for that the additivity-based sequence to reactivity algorithm both proteins. There was only a single band for rEPI1b, (Laskowski algorithm) originally developed and validated indicating high purity (Fig. 2B). The rEPI1a sample with typical Kazal domains might operate accurately for revealed two closely-migrating bands (Fig. 2B). The two atypical domains. bands reacted to the FLAG antibody and are likely to rep- resent rEPI1a with and without the signal peptide OMPA, Results which is located immediately before the FLAG peptide in The atypical Kazal domain of EPI1 is predicted to be a the vector pFLAG-ATS and is responsible for secreted functional inhibitor of subtilisin A expression in E. coli. Similar release of the mature protein The inhibition constants of two Kazal domains of EPI1 was commonly observed with other proteins expressed against subtilisin A were predicted using the Laskowski using pFLAG-ATS (M. Tian and S. Kamoun, unpublished). algorithm [12] based on the sequence of their 10 hyper- We also stained the gel loaded with purified rEPI1a pro- variable contact residues (Fig. 1). Interestingly, the atypi- tein with Coomassie blue. Compared with the band cor- cal Kazal domain EPI1a was predicted to be a strong responding to the rEPI1a without OMPA, the slower- inhibitor of subtilisin A with a Ki of 4.3 nM, a value that migrating band was much weaker (data not shown), indi- is remarkably similar to the experimentally determined Ki cating the secreted version of rEPI1a was the major com- of 2.77 +/- 1.07 nM for the entire EPI1 protein against ponent of the purified rEPI1a protein solution. Besides subtilisin A [7]. In contrast, the typical Kazal domain these two bands, no other proteins were detected by silver EPI1b, which contains the complete set of six cysteine res- staining suggesting that the rEPI1a preparation was highly idues, may not be functional against subtilisin A since the pure. predicted Ki was high at 50 mM. Therefore, these compu- tational analyses predicted that the atypical domain EPI1a is solely responsible for the inhibition of subtilisin A. Page 3 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 H Figure 2 eterologous expression of two Kazal domains of EPI1 Heterologous expression of two Kazal domains of EPI1. A, Amino acid sequences of recombinant Kazal domains rEPI1a and rEPI1b. The letters in upper case represent the amino acid sequence of the EPI1 protein. Residues in bold correspond to the native Kazal domains EPI1a and EPI1b as shown in Fig. 1. The letters in lower case represent the vector derived sequence with the underlined ones representing FLAG epitope tag. Numbers indicate the position of amino acid residues starting from the N terminus of EPI1 protein. B, Affinity purified recombinant Kazal domains visualized on SDS-PAGE stained with silver nitrate. The rEPI1a with the signal peptide OMPA is indicated by an asterisk. The numbers on the left represent the size of molecular weight markers. The atypical Kazal domain EPI1a inhibits the serine P69B, or A. tumefaciens containing the empty binary vec- protease subtilisin A tor pCB302-3. 10 µl of intercellular fluids from the two We performed inhibition assays of subtilisin A by incubat- treatments were used in in-gel protease assays. A distinct ing 0.2 µM of subtilisin A with 0.2 µM of rEPI1a, rEPI1b additional protease band was observed in the P69B- or buffer control in a volume of 50 µl. The remaining pro- expressing sample but not in the control suggesting that tease activity was measured using the QuantiCleave™ Pro- P69B-HA is functional (Fig. 4A). 10 µl of P69B-expressing tease Assay Kit as described in methods. In repeated N. benthamiana intercellular fluids were incubated with 20 assays, rEPI1a was found to inhibit about 91% of the pmol of the EPI1 recombinant protein rEPI1 [7], rEPI1a, measured activity of subtilisin A, whereas rEPI1b did not rEPI1b, or buffer and the remaining protease activity was display any significant inhibition (Fig. 3). These results detected by in-gel protease assay. rEPI1a, containing the are consistent with the prediction of the Laskowski algo- atypical Kazal domain, completely inhibited the P69B rithm (Fig. 1). It is unlikely that the FLAG tag interfered band similar to rEPI1. rEPI1 and rEPI1a also inhibited the with the inhibitory activities considering that both the activity of two other extracellular proteases from N. active rEPI1a and inactive rEPI1b carry the FLAG sequence benthamiana (Fig. 4B). The identity of these N. benthami- and that these experiments were conducted in parallel. ana proteases is unknown but they could also be subtili- sin-like serine proteases, such as homologs of tomato P69. EPI1a inhibits the tomato pathogenesis-related P69B In these experiments, rEPI1b did not exhibit any inhibi- subtilase tion towards P69B or other protease bands. EPI1 inhibits and interacts with the pathogenesis-related P69B subtilase of tomato [7]. To test which of the two EPI1a interacts with tomato P69 domains of EPI1 inhibits P69B, we first used agroinfiltra- We previously showed that rEPI1 interacts with P69B [7]. tion to transiently express P69B fused with the epitope tag Here, we tested whether the atypical Kazal domain EPI1a HA at the C-terminus in Nicotiana benthamiana leaves. interacts with P69B subtilase by coimmunoprecipitation. Intercellular fluids were collected from leaves infiltrated Coimmunoprecipitation was performed on BTH-induced with either Agrobacterium tumefaciens containing pCB302- tomato intercellular fluids incubated with rEPI1a, rEPI1b Page 4 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 The Figure 3 atypical Kazal domain EPI1a inhibits subtilisin A The atypical Kazal domain EPI1a inhibits subtilisin A. The remaining protease activity of subtilisin A was measured The atypical Ka Figure 4 zal domain rEPI1a inhibits P69B subtilase after incubating with rEPI1a, rEPI1b or without protease The atypical Kazal domain rEPI1a inhibits P69B sub- inhibitors (Std) using the QuantiCleave™ Protease Assay Kit tilase. A, In-gel protease assay of Nicotiana benthamiana as described in the methods. Activity is expressed as a per- intercellular fluids expressing the empty binary vector centage of total protease activity in the absence of protease pCB302-3 (-) or pCB-P69B (+). B, Inhibition assay of P69B by inhibitors. The bars correspond to the mean of three inde- recombinant EPI1 entire protein and the single Kazal pendent experiments with three replications for each experi- domains. P69B-expressing N. benthamiana intercellular fluids ment. The error bars represent the standard errors were incubated in the presence of rEPI1, rEPI1a, rEPI1b or calculated from the mean of three experiments. the absence of protease inhibitors (Buffer) and then the remaining protease activity was analyzed using zymogen in- gel protease assays. The arrow indicates the band location corresponding to the protease activity of P69B. or buffer control using FLAG antibody covalently linked agarose beads. Western blots were hybridized sequentially with P69 and FLAG antisera and revealed that P69 subti- lases co-precipitated with rEPI1a (Fig. 5). This indicates assays with varying concentrations of EPI1a (Fig. 6A). The that rEPI1a interacts with P69 subtilases. Since the P69 concentration of 0.15 µM of EPI1a resulted in inhibition family of subtilases have at least six homologs (P69A- levels of about 80% of the measured protease activity and P69F) [20,21] and the peptide used to generate the P69 was selected for the stability analysis. This concentration antisera is conserved among several homologs [7], we is within the linear part of the curve (Fig. 6A), suggesting cannot conclude that P69B subtilase is the only protein that hydrolysis of rEPI1a could be easily detected as a that was pulled down with rEPI1a. In contrast to rEPI1a, decrease in inhibitory activity. The inhibitory activity of rEPI1b could not be detected after coimmunoprecipita- rEPI1a did not show any decrease over 3 hours of incuba- tion with BTH-induced tomato intercellular fluids (Fig. tion with subtilisin A (Fig. 6B), indicating that EPI1a is a 5). However, rEPI1b was detected in control coimmuno- stable inhibitor of subtilisin A. These results are in sharp precipitations with the extraction buffer of tomato inter- contrast with those reported for domain LD-6 of LEKTI, cellular fluids (data not shown), suggesting that this which lost 50% of inhibitory activity after 1 hour of incu- protein is not stable in BTH-induced tomato intercellular bation with trypsin and lost all inhibitory effect after 3–4 fluids. hours [3]. EPI1a exhibits stable inhibitory activity Discussion To determine whether EPI1a is a temporary or stable Atypical Kazal domains with two disulfide bridges occur inhibitor of subtilisin, we performed stability analyses by in biologically important molecules. The 15-domain incubating subtilisin A with or without rEPI1a for increas- human serine protease inhibitor LEKTI that carries 13 ing periods of time and measuring the remaining protease atypical Kazal domains is associated with the severe con- activity. To determine the optimal concentration of EPI1a genital disorder Netherton syndrome [2]. The protease for the stability analyses, we first performed inhibition inhibitors EPI1 and EPI10 of P. infestans contain an Page 5 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 Coim a Figure 5 nd P6 munop 9 subtrilases using FLAG a ecipitation of the recomb ntiserainant Kazal domains Coimmunoprecipitation of the recombinant Kazal domains and P69 subtilases using FLAG antisera. Elu- ates from coimmunoprecipitation of rEPI1a, rEPI1b or buffter with proteins in BTH-treated tomato intercellular fluids were run on SDS-PAGE gel followed by sequential immunobloting with P69 (α-P69) and FLAG (α-FLAG) antisera at a dilution of 1:3000. atypical Kazal domain and have been implicated in viru- The atypical Kaz activity Figure 6 against subtilisin A al domain rEPI1a exhibits stable inhibitory lence of this devastating plant pathogen [7,19]. Although The atypical Kazal domain rEPI1a exhibits stable the structure and function of atypical Kazal domains from inhibitory activity against subtilisin A. A, Protease activ- LEKTI have been studied, the effects of the loss of Cys 3, ity of subtilisin A (0.2 µM) in the presence of rEPI1a in con- Cys 6 and the corresponding disulfide bond on inhibitor centrations ranging from 2 nM to 0.3 µM. Activity is expressed as a percentage of total protease activity in the reactivity and stability was not assessed and there is no absence of protease inhibitors. B, Protease activity of subtili- evidence showing that a single atypical Kazal domain can sin A (0.2 µM) after preincubation with 0.15 µM of rEPI1a be a stable inhibitor by itself [3,10,11]. In this study, we (black column) or without protease inhibitors (gray column) describe that the atypical domain EPI1a of the two- for a period of time ranging from 30 min to 180 min. Activity domain EPI1 protein is a stable inhibitor of the subtilisin is expressed as a percentage of total protease activity in the family of serine proteases. No loss of inhibitory activity absence of protease inhibitors at each treatment. The bars was found even after incubating EPI1a with subtilisin A correspond to the mean of three independent replications of for 3 hours. The loss of Cys 3, Cys 6 and the corresponding one representative experiment out of three performed. The disulfide bond does not have major adverse effects on error bars represent the standard errors calculated from the inhibitory activity or stability, indicating that these two three replications. cysteine residues might not be essential for the function of Kazal domains. This finding is important for determining the biochemical and biological functions of Kazal inhibi- tors containing atypical Kazal domain(s). Kazal-like pro- teins have been reported from animals, apicomplexans, atypical Kazal domains have been identified in plant oomycetes, as well as the bacterium Nitrosomonas europaea pathogenic oomycetes [7]. [7]. The availability of genomic sequence from a diversity of organisms is likely to reveal an increasing number of The structural mechanism underlying the stability of atypical Kazal inhibitors. For example, so far, a total of 14 EPI1a is not clear. The three-dimensional structure of the Page 6 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 atypical domain 6 (LD6) of LEKTI was determined. The total of 56 Kazal-like domains identified in five plant overall structure of LD6 resembles the three-dimensional pathogenic oomycetes have only four cysteines. Two of fold of typical Kazal-type inhibitors, but the backbone these Kazal-like inhibitors EPI1 and EPI10 of P. infestans geometry of its canonical loop is not well defined, provid- target the defense-related protease P69B of the host plant ing a possible explanation for its temporary inhibitory tomato. The first atypical Kazal domain of EPI1 appears to activity [11]. There are 13 residues between the first be solely functional in inhibiting and interacting with cysteine residue (Cys 1) and the second one (Cys 2) in P69B. In the three-domain EPI10, the second domain is LD6, instead of 6–9 in most typical Kazal domains [11]. also an atypical domain that was predicted to be func- The lack of one disulfide bond and the longer sequence tional against subtilisin based on the Laskowski algorithm stretch between the first two cysteines were proposed to be [19]. These findings raise some interesting questions. the factors responsible for the instability of LD6 [11]. What are the biochemical and biological implications of Indeed, the longer sequence stretch between the first two the loss of the disulfide bridge? Are there any evolutionary cysteines could explain the difference between EPI1a and advantages of the two-disulfide bridge Kazal domain over LD6. There are only 3 residues between Cys 1 and Cys 2 in the three-disulfide bridge domain in counteracting and EPI1a, which is shorter than in most Kazal domains. The co-evolving with host proteases? Additional functional longer sequence stretch of LD6 might lead to the abnor- and structural studies are needed to address these mal canonical loop and the non-permanent inhibitory questions. activity. Future work to determine the three-dimensional structure of EPI1a and compare it with LD6 of LEKTI and Conclusion other Kazal domains should help to unravel the structural In this study, the functionality of a two disulfide bridge mechanism underlying the functionality of atypical Kazal atypical Kazal domain EPI1a from Phytophthora was char- domains. acterized. EPI1a was predicted to be a strong inhibitor of subtilisin A using the additivity-based sequence to reactiv- The Laskowski algorithm was developed and validated ity algorithm (Laskowski algorithm). Inhibition assays based on typical Kazal domains [12,13]. The algorithm and coimmunoprecipitation experiments showed that exploits an exhaustive analysis of all amino acid variants recombinant domain EPI1a exhibited stable inhibitory in the ten hypervariable contact residues of turkey ovomu- activity against subilisin A and was solely responsible for coid third domain, a typical Kazal domain. The accuracy inhibition and interaction with tomato P69B subtilase, of the algorithm in predicting the reactivity of atypical providing evidence that the missing two cysteines and Kazal domains has not been evaluated (M. Laskowski Jr., their corresponding disulfide bond are not essential for pers. comm.). Here we found that the algorithm correctly inhibitor reactivity and stability. This report also suggests predicted which of the two EPI domains is likely to inhibit that the Laskowski algorithm originally developed and subtilisins. Our experimental data showed that the atypi- validated with typical Kazal domains might operate accu- cal Kazal domain EPI1a inhibited subtilisin A, and inhib- rately for atypical Kazal domains. ited and interacted with P69 subtilase similar to the entire EPI1 protein [7]. Also, using the algorithm, the atypical Methods Kazal domain EPI1a was predicted to be a strong inhibitor Prediction of inhibition constants of subtilisin A with a predicted Ki of 4.3 nM, which was in The putative ten hypervariable contact residues of EPI1a and EPI1b were identified based on similarity to canoni- very good agreement with the experimentally determined Ki of 2.77 +/- 1.07nM [7]. As expected from the predicted cal animal Kazal domains [14-16] and are shown in Fig. Ki of 50 mM, the typical EPI1b domain was not an effec- 1. Predicted inhibition constants for the EPI1 domains tive inhibitor of subtilisin A. In summary, it appears that against subtilisin A (Carlsberg) were generated by Drs. M. the Laskowski algorithm operates accurately for atypical A. Qasim and M. Laskowski Jr., Purdue University, with Kazal domains such as EPI1a. Perhaps, this is expected the additivity-based sequence to reactivity algorithm since the Cys 3 and Cys 6 residues of typical Kazal (Laskowski algorithm) described by Lu et al. [12]. domains are not contact positions. Nonetheless, our Plant growth and BTH treatment observations and the concordance between predicted and experimental data suggest that gross structural changes Tomato (Lycopersicon esculentum) cultivar Ohio 7814 and that could result from the loss of one disulfide bridge in N. benthamiana plants were grown in pots at 25°C, 60% atypical Kazal domains may not affect the specificity of humidity, under 16 hour-light/8 hour-dark cycle. We used the interactions between Kazal domains and their cognate the salicylic acid analog benzo-(1,2,3)-thiadiazole-7-car- serine proteases. bothioic acid S-methyl ester (BTH) to induce PR proteins. BTH treatment of tomato plants followed the exact same Atypical Kazal domains are ubiquitous in serine protease procedure described previously [7]. inhibitors of plant pathogenic oomycetes. Fourteen of a Page 7 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 Bacterial strains and plasmids ously [26]. A. tumefaciens strains carrying plasmids pCB- E. coli XL1-Blue and A. tumefaciens GV3101 were used in P69B, empty vector pCB302-3 [23], and pCB301-P19 (J. this study and were routinely grown in Luria-Bertani (LB) Win and S. Kamoun, unpublished) were used. pCB301- media [22] at 37°C and 28°C, respectively. Plasmids P19 is a construct expressing the P19 protein of tomato pFLAG-EPI1a and pFLAG-EPI1b for protein expression bushy stunt virus (TBSV), a suppressor of post-transcrip- were constructed by cloning the PCR amplified DNA frag- tional gene silencing in N. benthamiana that significantly ments corresponding to the coding sequence of Kazal enhances in planta transient expression [27]. Overnight domains EPI1a and EPI1b together with some flanking agrobacteria cultures were harvested by centrifugation at sequence into EcoRI and KpnI sites of pFLAG-ATS (Sigma, 2000 g for 20 min, and resuspended in 10 mM MgCl , 10 St. Louis, MO), a vector that allows secreted expression in mM MES (pH 5.6) and 150 µM acetosyringone. Resus- E. coli. The primers used for amplification of epi1a are pended agrobacteria cultures of pCB-P69B or pCB302-3 epi1a-F1(5'-gcggaattcTCAAAGCCCGCAAGTCATCAG-3') with an optical density (OD ) of 1.0 were mixed with and epi1a-R1(5'-gcgggtaccTTACTTGCTGGGAGGCT- equal volumes of a culture of pCB301-P19 with an optical GCTCGCCAG-3'). The primers used for amplification of density (OD ) of 2.0. The mixtures were kept at room CACCGGTAGCTCCACT- epi1b are epi1b-F1(5'-gcggaattc temperature for 3 hours and then infiltrated into leaves of GGCGAGCAGC-3') and epi1b-R1(5'-gcgggtaccTTATC- 6-week-old N. benthamiana plants. Intercellular fluids CCTCCTGCGGTGTC-3'). The introduced EcoRI and KpnI from infiltrated leaves were isolated 5 days after restriction sites for cloning are underlined. The letters in infiltration. upper case represent gene specific sequence. The detailed sequence information for the expressed fusion proteins Isolation of intercellular fluids FLAG-EPI1a (rEPI1a) and FLAG-EPI1b (rEPI1b) is shown Intercellular fluids were prepared from tomato and N. in Fig. 2A. Plasmid pCB-P69B is a construct with the open bethamiana leaves according to the method of de Wit and reading frame of the P69B gene [GenBank: Y17276] fused Spikman [28]. For tomato leaves, a 0.24 M sorbitol solu- with the HA tag (YPYDVPDYA) at the C-terminus cloned tion was used as extraction buffer. For leaves from N. into the binary vector pCB302-3 [23] and was described benthamiana, a solution of 300 mM NaCl, 50 mM NaPO elsewhere [19]. pH 7 [26] was used as extraction buffer. The intercellular fluids were filter sterilized (0.45 µM), and were used SDS-PAGE and Western blot analyses immediately or stored at -20°C. Proteins were subjected to 15% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) as previ- In-gel protease assays ously described [22]. Following electrophoresis, gels were In-gel protease assays were performed with 10% SDS- stained with silver nitrate following the method of Merril polyacrylamide gel containing 0.1% (w/v) gelatin (Bio- et al. [24] or with Coomassie Brilliant Blue [22], or the Rad Laboratories, Hercules, CA) using BIO-RAD's zymo- proteins were transferred to supported nitrocellulose gram buffer system as described earlier [7]. membranes (BioRad Laboratories, Hercules, CA) using a Mini Trans-Blot apparatus (BioRad Laboratories, Her- Inhibition assays of subtilisin A by EPI1 Kazal domains cules, CA). Detection of antigen-antibody complexes was Inhibition assays of subtilisin A by EPI1 Kazal domains carried out with a Western blot alkaline phosphatase kit were performed using colorimetric QuantiCleave™ Pro- (BioRad Laboratories, Hercules, CA). Antisera to P69 sub- tease Assay Kit (Pierce, Rockford, IL). 0.2 µM of subtilisin tilases were raised against a peptide specific for the tomato A (Carlsberg) (Sigma, St. Luis, MO) was preincubated P69 family [7]. Monoclonal anti-FLAG M2 antibody was with different amount of purified EPI1 Kazal domains, in purchased from Sigma (St. Louis, MO). a volume of 50 µl buffer for 30 min at 25°C, and then the remaining protease activity was measured following the Expression and purification of rEPI1a and rEPI1b procedures as described previously [7]. Analysis of the sta- Expression and purification of rEPI1a and rEPI1b was ble inhibitory activity of rEPI1a against subtilisin A was conducted as described previously for other pFLAG-ATS performed by incubating 0.2 µM of subtilisin A with 0.15 derived constructs [7,25]. Protein concentrations were µM of rEPI1a in 50 µl buffer (50 mM Tris, pH 8.0) for a determined using the BioRad protein assay (BioRad Labo- time period of 0–180 min at 25°C and then measuring ratories, Hercules, CA). To determine the purity, 0.5 µg of residue enzyme activity. the purified protein was run on a SDS-PAGE gel followed Coimmunoprecipitation by staining with silver nitrate. Coimmunoprecipitation of Kazal domains rEPI1a and Transient expression of P69B subtilase in planta rEPI1b with BTH-treated tomato intercellular fluids was Transient expression of P69B-HA in planta was performed performed using the FLAG-tagged protein immunoprecip- according to the agroinfiltration method described previ- itation kit (Sigma, St. Luis, MO) as described previously Page 8 of 9 (page number not for citation purposes) BMC Biochemistry 2005, 6:15 http://www.biomedcentral.com/1471-2091/6/15 T, Lin TY, Ogawa M, Otlewski J, Park SJ, Qasim S, Ranjbar M, Tashiro [7]. 100 pmol of purified rEPI1a or rEPI1b were preincu- M, Warne N, Whatley H, Wieczorek A, Wieczorek M, Wilusz T, bated with 300 µl of tomato intercellular fluids for 30 min Wynn R, Zhang W, Laskowski MJ: Predicting the reactivity of at 25°C. 40 µl of anti-FLAG M2 resin was added and incu- proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases. Proc Natl Acad Sci U S A 2001, bated at 4°C for 2 h with gentle shaking. The precipitated 98:1410-1415. protein complexes were eluted in 60 µl of FLAG peptide 13. Laskowski MJ, Qasim MA, Yi Z: Additivity-based prediction of equilibrium constants for some protein-protein associations. solution (150 ng/µl) and were analyzed by SDS-PAGE Curr Opin Struct Biol 2003, 13:130-139. and Western blot analyses. 14. Read RJ, Fujinaga M, Sielecki AR, James MN: Structure of the com- plex of Streptomyces griseus protease B and the third domain of the turkey ovomucoid inhibitor at 1.8-A Authors' contributions resolution. Biochemistry 1983, 22:4420-4433. MT, designing and performance of wet lab experiments, 15. Lu W, Apostol I, Qasim MA, Warne N, Wynn R, Zhang WL, Ander- writing of manuscript. SK, supervision of experimental son S, Chiang YW, Ogin E, Rothberg I, Ryan K, Laskowski MJ: Bind- ing of amino acid side-chains to S1 cavities of serine work, writing of manuscript. proteinases. J Mol Biol 1997, 266:441-461. 16. Laskowski MJ, Kato I, Ardelt W, Cook J, Denton A, Empie MW, Kohr WJ, Park SJ, Parks K, Schatzley BL, et al.: Ovomucoid third Acknowledgements domains from 100 avian species: isolation, sequences, and We are grateful to Dr. Michael Laskowski Jr. and Dr. M. A. Qasim from Pur- hypervariability of enzyme-inhibitor contact residues. Bio- due University for predicting the inhibition constants and sharing their chemistry 1987, 26:202-221. expert knowledge on Kazal inhibitors. We also thank Diane Kinney for 17. Qasim MA, Lu W, Lu SM, Ranjbar M, Yi Z, Chiang YW, Ryan K, Anderson S, Zhang W, Qasim S, Laskowski MJ: Testing of the addi- technical assistance and three anonymous reviewers for useful suggestions. tivity-based protein sequence to reactivity algorithm. Bio- This work was supported by USDA-NRI project OHO00963-SS. Salaries chemistry 2003, 42:6460-6466. and research support were provided by State and Federal Funds appropri- 18. Kamoun S: Molecular genetics of pathogenic oomycetes. ated to the Ohio Agricultural Research and Development Center, the Ohio Eukaryotic Cell 2003, 2:191-199. State University. 19. Tian M, Benedetti B, Kamoun S: A second Kazal-like protease inhibitor from Phytophthora infestans inhibits and interacts with the apoplastic pathogenesis-related protease P69B of References tomato. Plant Physiology 2005, 138:1785-1793. 1. Rawlings ND, Tolle DP, Barrett AJ: Evolutionary families of 20. Jorda L, Coego A, Conejero V, Vera P: A genomic cluster contain- peptidase inhibitors. Biochem J 2004, 378:705-716. ing four differentially regulated subtilisin-like processing pro- 2. Magert HJ, Kreutzmann P, Standker L, Walden M, Drogemuller K, tease genes is in tomato plants. J Biol Chem 1999, Forssmann WG: LEKTI: a multidomain serine proteinase 274:2360-2365. inhibitor with pathophysiological relevance. Int J Biochem Cell 21. 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Lu SM, Lu W, Qasim MA, Anderson S, Apostol I, Ardelt W, Bigler T, Chiang YW, Cook J, James MN, Kato I, Kelly C, Kohr W, Komiyama Page 9 of 9 (page number not for citation purposes)

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Published: Aug 23, 2005

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