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The crystal structures of two cuticle-degrading proteases from nematophagous fungi and their contribution to infection against nematodes Lianming Liang,*,†,1 Zhaohui Meng,*,†,‡,1 Fengping Ye,*,† Jinkui Yang,*,† Shuqun Liu,*,† Yuna Sun,§ Yu Guo,§ Qili Mi,*,† Xiaowei Huang,*,† Chenggang Zou,*,† Zihe Rao,§ Zhiyong Lou,§,2 and Ke-Qin Zhang*,†,2 *Laboratory for Conservation and Utilization of Bioresources and †Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, China; ‡Laboratory of Molecular Cardiology, Department of Cardiology, The First Affiliated Hospital of Kunming Medical University, Kunming, China; and §Tsinghua-Nankai-Institute of Biophysics Joint Research Group for Structural Biology, Tsinghua University, Beijing, China Cuticle-degrading proteases are involved in the breakdown of cuticle/eggshells of nematodes or insects, a hard physical barrier against fungal infections. Understanding the 3-dimensional structures of these proteins can provide crucial information for improving the effectiveness of these fungi in biocontrol applications, e.g., by targeted protein engineering. However, the structures of these proteases remain unknown. Here, we report the structures of two cuticledegrading proteases from two species of nematophagous fungi. The two structures were solved with X-ray crystallography to resolutions of 1.65 Å (Ver112) and 2.1 Å (PL646), respectively. Crystal structures of PL646 and Ver112 were found to be very similar to each other, and similar to that of proteinase K from another fungus Tritirachium album. Differences between the structures were found among residues of the substrate binding sites (S1 and S4). Experimental studies showed that the enzymes differed in hydrolytic activity to synthetic peptide substrates. Our analyses of the hydrophobic/ hydrophilic and electrostatic features of these two proteins suggest that their surfaces likely play important roles during fungal infection against nematodes. The two crystal structures provide a solid basis for investigating the relationship between structure and function of cuticle-degrading proteases.—Liang, L., Lou, Z., Ye, F., Yang, J., Liu, S., Sun, Y., Guo, Y., Mi, Q., Huang, X., Zou, C., Meng, Z., Rao, Z., Zhang, K.-Q. The crystal structures of two cuticle-degrading proteases from nematophagous fungi and their contribution to infection against nematodes. FASEB J. 24, 1391–1400 (2010). www.fasebj.org

ABSTRACT

Key Words: Ver112 䡠 PL646 䡠 serine protease 䡠 nematocidal virulence

Plant-parasitic nematodes, an important group of agricultural pests, have been reported to cause serious damages amounting to ⬎$100 billion/yr throughout 0892-6638/10/0024-1391 © FASEB

the world (1). Traditional control agents such as chemical nematicides and antithelminthic drugs have raised major environmental concerns and induced widespread resistance in field populations of many of these nematodes (2). As a result, nematophagous fungi, a group of natural enemies of nematodes, have attracted significant attentions because of their commercial potential for developing into effective biopesticides (3, 4). These fungi mostly live in the soil environment where nematodes are found. Among these fungi, some have trapping devices (such as adhesive branches, adhesive nets, constriction rings, etc.) to capture nematodes, some do not possess trapping devices but can secrete toxins to kill nematodes, and still others parasitize nematodes. It has been shown that these fungi play a crucial role in suppressing the populations of plant-parasitic nematodes in nature (4). Like most host-pathogen interactions, the following steps are involved in a typical nematode infection by nematophagous fungi: 1) recognition and adhesion of fungi to the cuticle of nematodes; 2) degradation and penetration of nematode cuticle/eggshells by enzymes secreted by fungi; and 3) immobilization and digestion of nematodes by fungi (5). Nematode cuticle, as well as insect cuticle (both ecdysozoans), are complex, composite structures with a high percentage of proteins and are the major barrier against fungal infection. The penetration of either the cuticle of nematodes/insects or their eggshells has been assumed to be the consequence of mechanical forces exerted by the fungi, in combination with cuticle-degrading enzymes (such as 1

These authors contributed equally to this work. Correspondence: K.-Q.Z., Laboratory for Conservation and Utilization of Bioresources, Yunnan University, 2 North Cuihu Road, Kunming 650091, China. E-mail: kqzhang111@yahoo. com.cn; Z.L., Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Tsinghua University, Beijing 100084, China; E-mail: [email protected] doi: 10.1096/fj.09-136408 2

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proteases, chitinases, and collagenases) produced by fungi. Of these enzymes, the proteases have been shown to play a critical role during host infection (2), and therefore have attracted particular attention (6). During the past several years, several cuticle-degrading proteases have been purified and characterized from different nematophagous or entomopathogenic fungi, including Arthrobotrys oligospora (7, 8), Pochonia chlamydospora (syn. Verticillium chlamydosporium) (9), Beauveria bassiana (10), and Metarhizium anisopliae (11). On the basis of their amino acid sequences, these cuticledegrading proteases have been identified as belonging to the serine protease S8 family (http://merops.sanger. ac.uk/). The nematophagous fungus Paecilomyces lilacinus has been widely studied and successfully implemented in controlling plant-parasitic nematodes (12–21). This fungus infects eggs and cyst nematodes using its secreted hydrolyzing enzymes (18, 22–24). In addition, several nematocidal toxins secreted by this fungus were also identified (25). In 1995, a basic serine protease pSP-3 was purified from P. lilacinus culture filtrate by affinity chromatography. Protease pSP-3 could be inhibited by phenylmethanesulfonyl fluoride (PMSF), and it shared a high degree of sequence similarity to subtilisin-like serine proteases (26). Incubation of the purified protease pSP-3 with nematode eggs significantly influenced their development (26). Transmission electron microscopic studies revealed that protease pSP-3 and chitinases from P. lilacinus drastically altered the eggshell structures when applied either individually or in combination (22). In the proteasetreated eggs, the inner lipid layer was disintegrated, and the middle chitin layer became thinner than in the negative controls (22). One way to improve the biocontrol potential of nematophagous fungi is to increase the pathogenicity by increasing the number of copies of cuticle-degrading genes in nematophagous fungi, using genetic engineering techniques. Recently, an A. oligospora mutant with additional copies of the PII gene was observed to develop a higher number of infection devices and showed an increase in the speed of capturing and killing nematodes when compared to the wild type (27). Another way is to change the key amino acid residues of protease or other virulence factors using site-directed mutagenesis. A fundamental issue with the success of this approach is to identify the key amino acid residues or domains associated with pathogenicity of proteases or other virulence factors. Structural studies of protease–inhibitor complexes can provide information on the binding modes of substrates and key amino acid residues associated with catalytic domains, as well as the mechanism of the hydrolysis reaction. In addition, some fungi, such as P. lilacinus, can infect both nematodes and insects, and opportunistically infect human patients (28 –30). Thus, the role of proteases during the interaction between nematophagous fungi and nematodes could be an ideal model to study the general roles of these proteases in fungi-host 1392

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interactions. Solving the structures of cuticle-degrading proteases will thus not only facilitate virulence improvements for fungi against nematodes or insects, but will also provide potential therapeutic drug targets against fungi in clinical treatment. However, no crystal structure of pathogenicity-associated proteases from this group of pathogenic fungi has been solved to date. In this article, we report the first two crystal structures of cuticle-degrading proteases, Ver112 and PL646, which were secreted by two nematophagous fungi, Lecanicillium psalliotae and P. lilacinus, respectively, and compared with proteinase K from a human parasitic fungus, Tritirachium album, both on catalytic properties and 3-D structure. These three fungi all belong to Ascomycota. Both L. psalliotae and P. lilacinus belong to the order Hypocreales. Hypocreales contains diverse groups of fungi, with some parasitic on insects, and others parasitic on other fungi. T. album belongs to the mitosporic Ascomycota, a heterogeneous group of ascomycotic fungi but sharing a common characteristic in that they do not have a known sexual state. Many human pathogenic fungi belong to the mitosporic Ascomycota.

MATERIALS AND METHODS Strains and plasmids The nematophagous fungi P. lilacinus (YMF1.00646) and L. psalliotae (YMF1.00112) were maintained in the Laboratory for Conservation and Utilization of Bioresources, Yunnan University. The nematode Caenorhabditis elegans was maintained in oatmeal medium (31). Protein purification The purification and crystallization of the proteases were described previously (32). Briefly, 10 L of culture filtrate was obtained by vacuum filtration, and the protein was concentrated through “salt out” using 56% (w/v) ammonium sulfate. The purification was performed on an AKTA explorer system (Amersham, Uppsala, Sweden). The sample was purified using a cation exchange chromatography column (Source 15S; 20 ml; Amersham). Fractions with protease activity were collected and then applied to a Superdex 75 (26/60) column (Amersham) equilibrated with 50 mM sodium phosphate buffer (PBS, pH7.5) containing 0.15 M NaCl and eluted with the same buffer. The elution peaks containing protease activity were collected and analyzed by SDS-PAGE. Protease activity and concentration were determined as described previously (33). The purity of the eluted protein was estimated by SDS-PAGE to be ⬎95%. Effects of temperature and pH on enzyme activity of PL646 The purified PL646 was mixed with the Britton-Robinson buffer system at pH values ranging from 5 to 12; and the protease activities were determined. Temperature effects were determined by incubating the reaction mixture at different temperatures (25– 80°C). Protease activities using casein as substrate were then measured using the method described previously (34).

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Effect of protease inhibitors on the enzyme activity of Ver112 and PL646 The purified PL646 and Ver112 were mixed with a serine protease-specific inhibitor, PMSF, and a tetrapeptide inhibitor, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MSU-AAPV-CMK), at various concentrations (0.1–1 mM). These mixtures were incubated under an optimal reaction condition for 30 min. Protease activities were then measured. Each treatment was repeated 3 times. Hydrolysis of cuticle proteins from nematodes by proteases Ver112 and PL646 and proteinase K Cuticle proteins of the nematode C. elegans were extracted using the method described by Cox et al. (35). One microliter (0.05 U; proteolytic unit as defined in ref. 33) purified Ver112, PL646, or proteinase K was mixed with 50 ␮l 0.2 M phosphate-citric acid buffer at pH values ranging from 3 to 12. An enzyme solution (50 ␮l) prepared as described above was then mixed with 50 ␮l cuticle proteins (0.2 mg) and incubated at 37°C for 30 min, and the cuticle proteins were then examined by SDS-PAGE. Moreover, the major bands of cuticle proteins were extracted and digested by trypsin (protease), followed by characterization of the digested products using liquid chromatography combined with mass spectrum analysis (LC-MS). The final peptide segments obtained were used as queries to search for related sequences using BLAST (www.ncbi.nlm.nih.gov/BLAST/) against the nematode-genome database, WormBase (http://www.wormbase.org/). Crystallization Two purified proteases were concentrated to 5 mg/ml in 10 mM PBS (pH 6.0) with 50 mM NaCl by using an amicon centrifugal filter device (Millipore, Bedford, MA, USA). Crystallization was performed by the hanging-drop vapor-diffusion method at 16°C. The optimal condition of crystallization was selected using crystal screen kits (Hampton Research, Laguna Niguel, CA, USA). Before crystallization, proteases Ver112 and PL646 were mixed with inhibitor MSU-AAPV ketone at a molar proportion of 1:5 and incubated at room temperature for 2 h. Then 1 ml of the protease or proteaseinhibitor solution was mixed with 1 ␮l of reservoir solution, and the mixture was equilibrated against 200 ␮l of reservoir solution at 16°C. The optimal reservoir solution for Ver112 contains 2 M ammonium sulfate and 5% isopropyl alcohol, while the optimal reservoir solution for PL646 contains 0.2 M potassium dihydrogen phosphate and 20% PEG 3350. Crystal formations were checked using a light microscope every 24 h. X-ray data collection, processing, and structure determination Data for the protease crystals were collected on a Rigaku R-AXIS IV⫹⫹ image plate with a Rigaku MM007 rotating anode home X-ray generator at 40 kV and 20 mA (␭⫽1.5418 Å; Rigaku, Tokyo, Japan). The crystals were soaked for several minutes in the reservoir solution supplemented with 20% glycerol as a cryoprotectant and then flash-cooled directly in liquid nitrogen. All data were collected at 100 K. Data were processed and scaled using the program HKL2000 (36). The structure of Ver112 was solved by molecular replacement using the program Phaser (37) with the proteinase K molecule (PDB code 1BJR) as an initial searching model. Two clear solutions in both the rotation and translation functions were found corresponding to two molecules in the asymmetric unit. Residues that differ between Ver112 and search CRYSTAL STRUCTURES OF PROTEASES VER112 AND PL646

model were manually rebuilt using the program O (38) under the guidance of Fo-Fc and 2Fo-Fc electron density maps. After refinement of the model with the CNS program (39) using simulated annealing, energy minimization, restrained individual B factors, and the addition of 274 water molecules, the respective working R factor and Rfree dropped from 0.45 and 0.42 to 0.22 and 0.25 for all data from 50.0 to 1.65 Å. The course of refinement was monitored by calculating Rfree based on a subset containing 5% of the total number of unique reflections. The final model of PL646 in complex with MSU-AAPV-CMK was also solved by molecular replacement based on the structure of proteinase K in complex with its inhibitor MSU-AAPA-CMK (PDB code 3PRK; ref. 40). After the same refinement steps and the addition of 517 water molecules, the working R factor and Rfree dropped from 0.45 and 0.48 to 0.23 and 0.26. The inhibitor, MSU-AAPV-CMK, was built by the guidance of Fo-Fc density map by using the program O (38). The quality of the final refined model was verified using the program PROCHECK (41). Final refinement statistics are summarized in Table 1. Structural representations were drawn with the program PyMol (42). Coordinates and structural factors have been deposited in the Protein Data Bank with accession codes 3F7M for Ver112 and 3F7O for PL646MSU-AAPV-CMK). Comparison of the structural and catalytic properties of PL646 and Ver112 with other related proteases The amino acid sequences of PRK and four typical cuticledegrading proteases [Pr1A (43), VCP1 (44), PL646, and Ver112], taken from the S8 family of the Merops peptidase database (http://merops.sanger.ac.uk/), were aligned using ClustalX 2.0 (45). Structural comparison was conducted using Swiss-PdbViewer (46). The active sites and substratebinding sites were compared using the programs PyMol (42) and CCP4mg (47). Several polypeptide substrates were designed and synthesized based on the sequence of the typical protease K inhibitor N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide through altering individual residues to check out the capacity and electrostatic properties of substrate-binding pockets S1 and S4 of the proteases. The kinetic parameters of proteases Ver112, PL646, and proteinase K toward the designed substrates were determined as described previously (48).

RESULTS Comparison of catalytic properties of Ver112, PL646, and proteinase K The protease PL646 was purified from P. lilacinus using gel filtration and ion-exchange chromatography. This enzyme showed the highest activity at 70°C and demonstrated reduced catalytic activity (from 100 to 47%) when temperature decreased from 70 to 40°C (Supplemental Fig. 1A). The optimal pH for the enzymatic catalysis is 10.0, with rapidly decreased activity at pH ⬍ 7.0 (Supplemental Fig. 1B). The optimal temperature and pH of PL646 are very similar to those of Ver112 (49). The hydrolytic activities of proteases Ver112 and PL646 were completely inhibited by low concentrations of PMSF (0.1 mM), and MSU-AAPV-CMK (0.1 mM). All soluble proteins of the nematode cuticle isolated from C. elegans could be digested by the enzyme Ver112 1393

TABLE 1.

Data collection and refinement statistics

Parameter

Data collection statistics Space group Unit-cell parameters (Å, deg) Resolution range (Å) Total reflections Unique reflections Redundancy Average I/␴(I ) Rmerge (%)a Data completeness (%) Matthews coefficient (Å3/Da) Assumed molecules in ASU Refinement statistics Reflections used (␴(F )⬎0) Rwork (%)b Rfree (%)b RMSD bond distance (Å) RMSD bond angle (deg) Average B value (Å2) Ramachandran plot (excluding Pro and Gly) Res. in most favored regions Res. in additionally allowed regions Res. in generously allowed regions

Ver112

PL646–MSU-AAPV

P212121 a ⫽ 43.7, b ⫽ 67.8, c ⫽ 76.3, ␣ ⫽ ␤ ⫽ ␥ ⫽ 90° 50.00–1.65 (1.71–1.60) 215,774 (8017) 26,819 (2077) 8.0 (3.8) 46.9 (14.0) 3.4 (13.8) 95.8 (75.4) 1.7 1

P21 a ⫽ 65.1, b ⫽ 62.5, c ⫽ 67.6, ␣ ⫽ ␥ ⫽ 90°, ␤ ⫽ 92.8° 50.0–2.2 (2.3–2.2) 110,259 (8026) 27,780 (2685) 4.0 (3.4) 12.1 (3.2) 13.1 (46.0) 98.9 (96.4) 2.1 2

21,764 21.4 25.1 0.004 1.262 17.1

26,467 23.2 26.3 0.009 1.621 22.8

89.3% 10.3% 0.4%

88.0% 11.3% 0.7%

Numbers in parentheses are corresponding values for the highest-resolution shell (2.5⫺2.4 Å). aRmerge ⫽ 冱 h 冱 l 兩Iih ⫺ ⬍Ih⬎兩/冱 h 冱 I⬍Ih⬎, where ⬍Ih⬎ is the mean of multiple observations Iih of a given reflection hour. bRwork ⫽ 冱 㛳Fp(obs)兩 ⫺ 兩Fp(calc)㛳/冱 兩Fp(obs)兩; Rfree is an R factor for a selected subset (5%) of reflections that was not included in prior refinement calculations.

at pH values from 6 to 12. However, the catalytic activity was lower at pH values ranging from 3 to 5, resulting in incomplete hydrolysis of the cuticle proteins (Fig. 1). Similar results were obtained when nematode cuticle proteins were mixed with PL646 and proteinase K (see Supplemental Fig. 2). The cuticle peptide segments obtained from the LC-MS assays were further analyzed by subjecting them to BLAST searches against the database “C. elegans (WS190) proteins” at the WormBase Web site. The results showed that these peptide segments had significant similarity to the following proteins: myosin class II heavy chain, basement membrane-specific heparan sulfate proteoglycan (HSPG)

core protein, mitochondrial inner membrane protein, actin, and F0F1-type ATP synthase ␣ and ␤ subunits (see Supplemental Table 1). PL646 can also hydrolyze these cuticle proteins under neutral and alkaline conditions. Pure collagen (Type I⬘; Worthington Biochemical, Lakewood, NJ, USA) was used to examine the collagendegrading capacity of three proteases, as described previously (49). They showed similar catalytic capacities against collagen: Ver112, 14%; PL646, 10%; and proteinase K, 14% of the activity of each protease against casein. Crystal structures of the proteases

Figure 1. Ver112 hydrolyzes nematode cuticle proteins. All protein components in the nematode cuticle were hydrolyzed by the enzyme Ver112 at pH ⬎ 6, but at pH 3–5, catalytic activity was lower, resulting in incomplete hydrolysis of the cuticle protein components. Ver112 bond in each column is marked. Lanes: Ver112, protease Ver112 only; numbers indicate pH of each reaction. 1394

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The structures of both Ver112 and PL646 consist of 6 ␣ helices, a 9-stranded parallel ␤ sheet, and three 2-stranded antiparallel ␤ sheets (Fig. 2A, B). The substrate-binding site of Ver112 includes two peptide segments containing residues 103–107 and 135–139, respectively; the corresponding segments in PL646 are residues 102–106 and 134 –138, respectively. The catalytic triad of Ver112 is composed of residues Asp41, His72, and Ser-227 (Ser-225 in PL646). There are 5 cysteines in both proteases. In Ver112, two disulfide bridges are formed by residues Cys36 –Cys125 and Cys180 –Cys251, whereas the residue Cys75 is free. In PL646, residues Cys36 –Cys126 and Cys181–Cys253 form two disulfide bridges, and Cys76 is free. Many proteases in the proteinase K family contain Ca2⫹-binding sites. It has been observed that Ca2⫹

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the proteinase K complexed with MSU-AAPA ketone (PDB code 3PRK; ref. 40), exhibiting a superimposed backbone RMSD of 0.69 Å. As shown in Fig. 3, MSUAAPV ketone is located in a cleft formed between the two peptide segments containing residues 102–106 and 134 –138, resulting in the formation of an antiparallel 3-stranded pleated sheet. Five hydrogen bonds were observed within this ␤ sheet. They are formed between the carbonyl oxygen (R, receptor) of the MSU group and N of Tyr107 (D, donor); the N atom of Ala (P4, D) and carbonyl oxygen (R) of Gly105; the carbonyl oxygen of Ala (P4, R) and the N atom (D) of Gly105; the N atom of Ala (P3, D) and carbonyl oxygen (R) of Gly137; the carbonyl oxygen of Ala (P3, R) and the N atom (D) of Gly137; the N atom of Val (P1, D) and the carbonyl oxygen (R) of Ser-135. The carbonyl oxygen of Val (P1) contacts both the ␦N atom of Asn164 and the amido N atom of Ser-227, participating in the formation of the oxyanion hole. The active site residues His72 and Ser-227 react with the terminal-COCH2C1 group, forming two covalent bonds with PL646, i.e., the bonds between the C atom of Val (P1) and O␥ atom of Ser-227 and between the C-terminal carbon atom and εN atom of His72. The electrostatic surface potentials of the proteases were further analyzed using APBS (52) and PyMol. Figure 2D, E shows that the substrate-binding region (including the active center) is primarily negatively charged in both Ver112 and PL646. In contrast, the surface opposite the substrate-binding region is mainly positively charged (Fig. 2F, G). These common electrostatic features between the two cuticle-degrading enzymes may contribute to their common infection characteristics against nematodes. Comparison on the structures and catalytic properties of related proteases Figure 2. Overall structures of Ver112 and PL646. A, B) Ribbon models of Ver112 (A) and PL646 (B); Ca2⫹ location in PL646 is indicated. C) Ca2⫹ binding site overlapped with a 2Fc-Fc electron density map contoured at 1.0 ␴. Ca2⫹ is bound with a high affinity by carbonyl oxygens of Glu177, Val180, and Leu201, and O␥1 of Thr182 and O␦2 Asp203. D–G) Electrostatic surface potential of Ver112 (D, F) and PL646 (E, G). Locations of the substrate-binding pockets, S1–S4, are indicated. Positive and negative surface potentials are contoured blue and red, respectively. Negatively charged area around the substrate-bonding site is encircled by a cyan line.

enhances the thermal stability of proteases (50, 51). There is only one Ca2⫹-binding site in PL646, with the Ca2⫹ being bound with high affinity by carbonyl oxygens of Glu177, Val180, and Leu201, O␥1 of Thr182, and O␦2 Asp203 (Fig. 2C). For Ver112, although the Ca2⫹-binding residues are identical at the same positions to those in PL646, no Ca2⫹ was observed to bind to the protein. The possible reason for this is that Ver112 was crystallized in a Ca2⫹-free buffer. The structure of protease PL646 in complex with the inhibitor MSU-AAPV ketone is very similar to that of CRYSTAL STRUCTURES OF PROTEASES VER112 AND PL646

Fig. 4A shows the amino acid sequence alignment of 5 cuticle-degrading proteases. A high degree of sequence identity was observed between these enzymes derived from different pathogenic fungi. For example, the sequence identities of PL646 with Pr1A, VCP1, Ver112, and PRK are 76.2, 69.0, 72.6, and 62.5%, respectively. As expected, the amino acid residues within the catalytic triad, substrate binding sites, and Ca2⫹-binding site are highly conserved. The high degree of sequence similarity between Ver112 and PL646 likely contributed to the nearly identical structures of the two enzymes. On the basis of the information above, we expect that the structures of the other cuticle-degrading proteases may also be very similar. Some amino acid differences were observed in the substrate binding sites among the five proteases. For example, for the S1 pocket, residue at position 165 (PL646 numbering) is Asn in PRK, whereas in the other four cuticle-degrading proteases it is Asp. A close comparison of the structures of PRK, PL646, and Ver112 revealed that the conformations adopted by Asn and Asp were similar, with their side chains pointing 1395

Figure 3. Substrate-binding site of PL646 bonding the inhibitor MSU-AAPV-ketone. A) MSUAAPV ketone is located in the cleft formed between the two peptide segments 102–106 and 134 –138. C terminus is covalently bonded to the catalytic center residues His72 and Ser-227; carbonyl oxygen of P1 Val is located at the oxyanion hole consisting of the ␦ N atom of Asn164 and amino N atom of Ser-227. Additional 6 H bonds were observed between PL646 and the inhibitor. B) Schematic drawing and atom numbering of the complex as described in A. Oxyanion hole is indicated; scissors indicate the cleavage bond.

inside the S1 pocket (Fig. 4C). The Asp in the cuticledegrading proteases contributes more anionic characteristics to the S1 site than Asn; and such an electrostatic feature might have functional relevance (53). More residue alterations are observed in the S4 pocket, i.e., residues at positions 106, 107, 110, 139, and 144 (PL646 numbering) are different among these enzymes. Specifically, residue Gln106 in PRK is replaced by Ser in the other four proteases. Although both the Asn and Ser have polar side chains, the side chain of Ser-106 in the cuticle-degrading proteases is smaller than that of Gln, leading to a larger S4 pocket in cuticle-degrading proteases than that in PRK. Furthermore, the smaller hydrophobic residue, Leu at position 107 in Ver112, compared to Tyr107 in PL646, could result in a larger S4 pocket in Ver112 than in PL646. The residue at position 110 is a Val in VCP1, which is replaced by Ile in the other four proteases. The side chain of Ile is larger than that of Val; therefore, the size of S4 pocket are larger in VCP1 than in other enzymes. The residue 139 is a Pro in VCP1, which is more hydrophobic than Gly in the other four proteases, contributing more hydrophobic property to the VCP1 S4 pocket. At position 144, the side chain of Leu in Ver112 is larger than that of Val in the other four proteases. Moreover, both residues are located at the bottom of S4 pocket; therefore, the relatively larger Leu makes S4 pocket of Ver112 flatter than that of the rest proteases (Fig. 4C). The multiple sequence alignment (Fig. 4A) also reveals 5 cysteines common to the 5 proteases. These cysteines form two disulfide bridges, Cys36-Cys125 and Cys180-Cys251 (PL646 numbering) in the crystal structures of PRK, Ver112, and PL646 except for the Cys75 that remain free. Because of the high degree of sequence identity of these enzymes (which should lead to nearly identical structure), we can assume that the two disulfide bridges should also exist in structures of the other two cuticle-degrading proteases, VCP1 and Pr1A. The disulfide bridge Cys36-Cys125 links ␤3 and a loop located between ␤6 and ␣3, contributing to the stability of the region close to the catalytic residue Asp39 and of 1396

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the nearby substrate-binding sites such as S1 (53). The other disulfide, Cys180-Cys251, which links ␤8 and ␣6, stabilizes the C-terminal region of the enzymes. The three currently known structures of serine proteases, Ver112, PL646, and PRK (PDB code 3PRK) were superimposed using their backbone carbon atoms. Figure 4B shows that the backbone folds of the three enzymes are essentially identical, with the exception of some surface exposed regions such as N- and C-termini (Fig. 4B, i), the loops located between ␤4 and ␣3 (Fig. 4B, ii), and between ␣6 and ␣7 (Fig. 4B, iii). We speculate that it is the insertions and/or deletions of residues in these regions that cause relatively large conformational differences. However, the overall pairwise C␣ RMSDs of PL646-Ver112, PL646 –3PRK, and Ver112– 3PRK are small; 0.57, 0.69, and 0.70 Å, respectively. Kinetics analysis of Ver112, PL646, and proteinase K To compare the catalytic properties of the three proteases, several substrates were designed based on a previously reported substrate, Suc-AAPF-pNA (48). For the canonical proteinase K (PRK), the strongest substrate affinity occurred toward AAPD due to the lowest Km value, as shown in Table 2. However, the cuticledegrading enzyme PL646 showed relatively weak affinity toward AAPD, and this substrate may not bind to Ver112, as the kinetic parameters could not be determined. We speculate that it is the Asp165 in the S1 pocket that hinders (in PL646) or abolishes (in Ver112) AAPD binding because of the electrostatic repulsion. However, our assay could not obtain the kinetic parameters of PRK toward NAPF, likely caused by the relatively small S4 pocket of PRK when compared to that of Ver112 and PL646. Ver112 and PL646 both show high affinity toward substrates NAPF and PAPF, reflecting their capability of accommodating peptide substrate with large P1 and P4 residues. Interestingly, Ver112 binds with slightly higher affinity than does PL646 to NAPF and PAPF, which is probably caused by a larger S4 pocket in Ver112 than in PL646, as described above.

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Figure 4. Structural and sequence comparisons of homologue proteases. A) Multiple sequence alignment of PL646 (ABO322 56), Ver112 (AAU01968), VCP1 (CAD20580), Pr1A (P29138), and PRK (P068 73). Secondary structure elements are marked on top of the alignment; ␣-helices and ␤-strands are presented as curves and arrows, respectively. Residues belonging to the S1 site are circled by green frames; residues belonging to the S4 site are circled by blue frames; ⬃ indicates residues involved in both the S1 and S4 sites. Residues forming the catalytic triad (Asp, His and Ser,) are marked with blue triangles. Cysteines involved in the disulfide bridge are marked with red triangles. B) Superposition of the structures of Ver112 (magenta), PL646 (cyan), and proteinase K (yellow). Structures of the three enzymes are essentially identical, except for some surface-exposed loops such as the N and C termini (i), and those located between ␤4 and ␣3 (ii), and between ␣6 and ␣7 (iii). C) Differences in residues in the S1 and S4 pockets between Ver112 (magenta), PL646 (cyan), and PRK (yellow). Positions involved in amino acid variations are labeled (PL646 numbering); corresponding residues are shown as sticks.

In addition, the relatively smaller S4 pocket in PL646 may cause difficulty in stereochemical rearrangement when interacting with P1 Ala of AAPF and AAPD, thereby leading to PL646’s relatively weaker affinity toward these two substrates than Ver112. Interestingly, although PL646 showed the lowest affinity toward AAPF, its high turnover rate (170/s) counteracted this effect leading, in turn, to the highest catalytic efficiency (Kcat/Km), which was 1 or 2 orders of magnitude larger than that toward AAPD, NAPF, or PAPF (see Table 2). Similar results were also observed from Ver112 and PRK, confirming AAPF as the most suitable substrate CRYSTAL STRUCTURES OF PROTEASES VER112 AND PL646

for enzyme activity assay. The relatively lower turnover rates of these three enzymes toward PAPF, NAPF, and AAPD may arise from the stereochemical constraints, while interacting with the large P4 (such as P and N in PAPF and NAPF, respectively) and P1 residues (such as D in AAPD). For the three enzymes assayed here, PAPF was the most optimal substrate among these three newly designed substrates because of its high affinity and turnover rate. Our results are in agreement with previously reported data (40), reflecting that the proteinase K-like cuticle-degrading proteases also have broad substrate specificity with slight preference for bulky hydrophobic 1397

TABLE 2.

Kinetics analysis of Ver112, PL646, and proteinase K against synthesized substrates AAPF

AAPD

NAPF

PAPF

Enzyme

Km

Kcat/Km

Km

Kcat/Km

Km

Kcat/Km

Km

Kcat/Km

Ver112 PL646 PRK

0.145 1.42 0.31

27354 17957 33142

NA 1.21 0.093

NA 166 1229

0.164 0.695 NA

2067 453 NA

0.226 0.472 0.111

5206 1576 3558

AAPF, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; AAPD, N-succinyl-Ala-Ala-Pro-Asp-p-nitroanilide; NAPF, N-succinyl-Asn-Ala-Pro-Phe-p-nitroanilide; PAPF, N-succinyl-Pro-Ala-Pro-Phe-p-nitroanilide; NA, not available (kinetics parameters cannot be determined).

or aromatic residues at the P1 and P4 positions of the substrate.

DISCUSSION In this study, two cuticle-degrading proteases, Ver112 and PL646, were isolated from nematophagous fungi L. psalliotae and P. lilacinus, and their crystal structures were determined. To our knowledge, this is the first report on the 3-D structure of cuticle-degrading proteases from nematophagous fungi. Because of the high degree of sequence similarity between Ver112 and PL646 and with other members of the serine proteases family S8, the two proteases should belong to this group of enzymes (Fig. 4A). Many proteases within the S8 family are produced by parasite fungi such as A. oligospora (7, 8), P. chlamydospora (9), B. bassiana (10), and M. anisopliae (11). These proteases are believed to play important roles in infection against hosts. Both Ver112 and PL646 showed high hydrolytic activities against cuticle proteins derived from C. elegans and synthesized proteinous substrates at broad ranges of temperature (4 –70°C) and pH (7.0 –10.0), with the highest enzymatic activity observed at 70°C and pH 10. Other proteases derived from nematophagous or entomoparasitic fungi were also reported to have high activities at high temperature and alkaline condition (2). The high temperature may enhance conformational flexibility of enzyme structures, especially in the substrate-binding regions, thus enhancing the substrate affinity and catalytic efficiency of these enzymes (52). However, excessively high temperatures can also lead to thermal denaturation of the proteases, and therefore, the temperature just below the transition/denaturation temperature is often the optimal reaction temperature of these proteases. The currently reported cuticledegrading proteases are all alkaline proteases due to the relatively high pH value (pH 8 –10) required for their optimal catalytic reaction (54). A high pH value guarantees the correct protonation state for one of the catalytic triad residues, His (i.e., protonation on the N␦1 but not on Nε2 atom of the imidazole group in His), which, in turn, maintains the correct hydrogen bonding within the catalytic triad (i.e., the hydrogen bonds His-N␦1-H…O␦2-Asp and Ser-O␥-H…Nε2-His) and the correct proton transfer. Moreover, high pI of these alkaline proteases can help maintain the large 1398

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patches of positively charged surfaces of the enzyme molecules in natural environment (Fig. 3D, G), enhancing the capacity of the cuticle-degrading proteases to adsorb to cuticles carrying abundant acidic charge (52, 55–57). The crystal structures of PRK, Ver112, and PL646, in conjunction with the multiple sequence alignment of the cuticle-degrading proteases, allowed us to infer the substrate specificities of these proteases at the amino acid level. The substrate-binding pockets within both enzymes are large and in the case of S1, hydrophobic. Therefore, the P1 substrate residues with bulky hydrophobic side chains (such as phenylalanine) are favored by the S1 pocket. Although the S4 pocket is smaller than the S1 pocket, it is still large enough to accommodate large P4 substrate residues, a fact that is reflected by the lower Km values of PL646 toward PAPF and NAPF than toward AAPD and AAPF. The possible reasons for this include that more bonding contacts (such as hydrogen bonding, hydrophobic and van der Waals interactions) can be formed between large P4 residues and S4 pocket than between small P4 residues and S4 pocket. Moreover, we also found that the cuticle-degrading proteases investigated here have a tyrosine (Tyr107) in the S4 pocket with the exception of Ver112. The side chain of the Tyr, which is a large phenoxyl ring, is located in the external region of the S4 pocket and acts as a lid to this pocket, thereby restricting to a certain extent the binding of the peptide substrate with large P4 side chains (58, 59). The results of our kinetics analyses, i.e., the reduced affinities of PL646 toward NAPF and PAPF in comparison with those of Ver112, support this hypothesis. The electrostatic surface potentials of the two cuticledegrading proteases, Ver112 and PL646, demonstrate a common feature of these two enzymes: the negatively charged surface patches are mainly concentrated on the substrate-binding regions, while the positively charged patches are located on the remaining surfaces of the molecules. It has been suggested that the anionic feature of the substrate-binding regions would increase the local conformational flexibility and enhance catalytic efficiency (52). As mentioned above, the large positively charged areas on most of the molecular surface can increase the adsorption of the cuticle-degrading proteases to cuticles bearing abundant acidic residues. Thus, it seems likely that such an electrostatic surface feature of these cuticle-

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LIANG ET AL.

degrading proteases contributes significantly to the infection against nematodes. The degradation of nematode cuticle/exoskeleton is a key step for infection by nematophagous fungi. In this study, all the proteinous components extracted from C. elegans were completely degraded by Ver112 (Fig. 1) and PL646, suggesting the crucial role of these two proteases in the infection process. Moreover, the broad substrate specificity of cuticle-degrading proteases could possibly allow these fungi to maximally utilize different kinds of cuticle/exoskeleton proteinous components to obtain nutrients. Equal units of proteinase K from T. album (a human pathogen fungus) showed similar hydrolytic activity against soluble proteins of nematode cuticle and pure collagen, which suggests that during fungal infection, proteinase K, Ver112, and PL646 might work by similar mechanisms. The nematocidal/insecticidal activity of fungi may be improved by protein engineering of the cuticle-degrading protease profiting from the two crystal structures obtained here. Meanwhile, our efforts to determine crystal structures of the two proteases and compare their molecular features (e.g., hydrophobic/hydrophilic and electrostatic features of substrate binding sites) may also aid in designing more potent inhibitors against these proteases, facilitating, in turn, the therapeutic treatments for diseases caused by human pathogenic fungi. The authors are grateful to Prof. Jianping Xu (Department of Biology, McMaster University, Hamilton, ON, Canada), for valuable comments and critical discussion. This work was funded by the National Basic Research Program of China (2007CB411600), by projects from the National Natural Science Foundation of China (30630003, 30660107, 30960229, 30860011, 30860278, and 30221003), and the Department of Science and Technology of Yunnan Province (2007C007Z, 2006C0071M, 2007C0001R, and 2009CI052).

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Received for publication May 7, 2009. Accepted for publication November 12, 2009.

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