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The crystal structure of the secreted aspartic protease 1 from Candida parapsilosisin complex with pepstatin A

Jirí Dostál a,b, Jirí Brynda b,c, Olga Hrušková-Heidingsfeldová a,b, Irena Sieglová b,c,Iva Pichová a,b, Pavlína Rezácová b,c,*

a Gilead Sciences and IOCB Research Centre, Flemingovo námestí 2, 166 10 Prague 6, Czech Republicb Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo námestí 2, 166 10 Prague 6, Czech Republicc Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo námestí 2, 166 10 Prague 6, Czech Republic

a r t i c l e i n f o

Article history:Received 16 January 2009Received in revised form 18 March 2009Accepted 17 April 2009Available online 3 May 2009

Keywords:Candida parapsilosisPepstatin ASecreted aspartic proteaseCrystal structure

a b s t r a c t

Opportunistic pathogens of the genus Candida cause infections representing a major threat to long-termsurvival of immunocompromised patients. Virulence of the Candida pathogens is enhanced by productionof extracellular proteolytic enzymes and secreted aspartic proteases (Saps) are therefore studied aspotential virulence factors and possible targets for therapeutic drug design. Candida parapsilosis is lessinvasive than C. albicans, however, it is one of the leading causative agents of yeast infections.

We report three-dimensional crystal structure of Sapp1p from C. parapsilosis in complex with pepstatinA, the classical inhibitor of aspartic proteases. The structure of Sapp1p was determined from protein iso-lated from its natural source and represents the first structure of Sap from C. parapsilosis. Overall fold andtopology of Sapp1p is very similar to the archetypic fold of monomeric aspartic protease family andknown structures of Sap isoenzymes from C. albicans and Sapt1p from C. tropicalis. Structural comparisonrevealed noticeable differences in the structure of loops surrounding the active site. This resulted in dif-ferential character, shape, and size of the substrate binding site explaining divergent substrate specific-ities and inhibitor affinities. Determination of structures of Sap isoenzymes from various species mightcontribute to the development of new Sap-specific inhibitors.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

The yeasts of the genus Candida represent a predominant causeof fungal infections in humans (Pfaller and Diekema, 2007). Patho-genic Candida species are commensal organisms in healthy individ-uals, but when the immunity of the host is reduced, Candidacolonization can lead to an infection. Depending on the immunestatus of the host, Candida spp. can cause relatively mild skinmycoses, but also life-threatening invasive infections. Since manyantifungal drugs have been marketed, most cases of superficialmycoses can be managed. However, the number of suitable thera-peutic strategies for invasive candidiasis is limited, and thereforesystemic Candida infections in immunocompromised patients areassociated with high mortality rates (Weig and Brown, 2007).

Although C. albicans is the most frequently encountered Candidain clinical practice, the incidence and clinical significance of non-albicans species have increased in the past several years (Laiet al., 2008; Pfaller and Diekema, 2007). The reported incidence

of non-albicans species varies considerably among the geographicalareas and patient groups. Nevertheless, C. parapsilosis ranks as thesecond or the third most common cause of Candida bloodstreaminfections in studies performed in Europe, Canada, and Latin Amer-ica (Caggiano et al., 2008; Colombo et al., 2006; Costa-de-Oliveiraet al., 2008; Odds et al., 2007; Pfaller et al., 2008). C. parapsilosiswas found to be less invasive than C. albicans (Jayatilake et al.,2006). Despite this, it is one of the leading causative agents of nos-ocomial infections by yeasts, and especially threatens the patientsat neonatal intensive care units (Trofa et al., 2008).

To penetrate host tissues, pathogenic Candida species employextracellular hydrolytic enzymes, namely aspartic proteases, li-pases, and phospholipases (Haynes, 2001; Schaller et al., 2005).These hydrolases usually cluster in gene/protein families, and theexpression of individual isoenzymes thus can be differentially reg-ulated depending on environmental conditions, such as pH, tem-perature or a type of nutrient sources. While the secretedaspartic proteases of C. albicans have been extensively studied inmany laboratories (reviewed in Hruskova-Heidingsfeldova, 2008),information on orthologous enzymes from non-albicans Candidaspp. is limited.

In C. parapsilosis, three genes encoding secreted aspartic prote-ases have been identified and denominated SAPP1–3. While the

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* Corresponding author. Address: Institute of Organic Chemistry and Biochem-istry, Academy of Sciences of the Czech Republic, Flemingovo námestí 2, 166 10Prague 6, Czech Republic. Fax: +420 220 183 280.

E-mail address: rezacova@uochb.cas.cz (P. Rezácová).

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SAPP3 gene product remains enigmatic, the Sapp1p and Sapp2pisoenzymes have already been subjected to enzymological studies(Dostal et al., 2005; Merkerova et al., 2006). Both of these enzymeshave broad substrate specificity, but they differ in catalytic activi-ties and expression pattern. While the SAPP2 gene appeared to betranscribed constitutively, SAPP1 transcription is induced by thepresence of an exogenous protein as the sole source of nitrogen(Hruskova-Heidingsfeldova, 2009).

The essential role of secreted aspartic proteases in virulence dur-ing local and systemic Candida spp. infections (Cutler, 1991) makesthese enzymes an attractive target for therapeutic drug design. Theknowledge of three-dimensional structures of various Sap isoen-zymes from different species is crucial to assist the Sap-specificinhibitor design. The crystal structures of four Sap isoenzymesSap1, 2, 3, and 5 from C. albicans are available to date (Abad-Zapateroet al., 1996, 1998; Borelli et al., 2007, 2008; Cutfield et al., 1995), butnot many Sap enzymes from non-albicans species have been charac-terized on a structural level. In fact, the only crystal structure avail-able is the Sapt1p from C. tropicalis (Symersky et al., 1997).

Here we report the first X-ray structure of secreted aspartic pro-tease Sapp1p from C. parapsilosis in complex with pepstatin A at1.9 Å resolution. We took advantage of the inducibility of the SAPP1gene and purified an amount of Sapp1p sufficient for structuralstudies directly from the C. parapsilosis culture media. We comparedthe Sapp1p structure with the known structures of orthologous pro-teins Sap2 and Sapt1p from C. albicans, and C. tropicalis, respectively.Interactions with the inhibitor were analyzed and compared toavailable structures of Sap enzymes–pepstatin A complexes.

2. Experimental

2.1. Strains and growth conditions

Candida parapsilosis strain P-69 was obtained from the mycolog-ical collection of the Faculty of Medicine, Palacky University, Olo-mouc, Czech Republic. The yeasts were cultivated in YCB-BSAmedium (1.2% (w/v) yeast carbon base, 0.2% (w/v) BSA, 15 mM so-dium citrate, pH 4.0).

2.2. Purification of Sapp1p

Sapp1p was purified from the cell-free supernatants obtainedfrom C. parapsilosis cultivation in the YCB-BSA medium pH 4.0,for 72 h at 30 �C in a rotation shaker. The cells were harvested bycentrifugation (5000g for 15 min). The supernatant was filteredusing Stericup (Millipore), and the cell-free supernatant containingsecreted proteases was precipitated by addition of ammonium sul-fate (final concentration 80% (w/v)) and centrifuged at 20 000g for10 min. The sediment was diluted in 15 mM sodium citrate, pH3.75, and dialyzed against the same buffer in order to removeammonium sulfate. Sapp1p was purified by anion-exchange chro-matography using an FPLC equipped with the MonoS (Amersham)column equilibrated in 15 mM sodium citrate, pH 3.75. Elution wascarried out using a NaCl gradient (0–300 mM). Samples containingactive Sapp1p were dialyzed into the 25 mM bis–Tris, pH 6.3, andfurther purified using the MonoP HR 5/20 (Amersham) column.Sapp1p elution was performed using gradient of 10% (v/v) polybuf-fer 74, pH 4.0 (Amersham). The efficiency of purification steps wasanalyzed using SDS–PAGE and activity assays.

2.3. Protein analysis

Identity of Sapp1p was confirmed using peptide mass finger-printing and N-terminal sequencing. Size-exclusion chromatogra-phy was used to analyze the quality of the protein sample.

2.3.1. Peptide mass fingerprintingSapp1p was excised from the gels and was in-gel digested with

trypsin. Extracted peptides were concentrated in SpeedVac, (Ther-mo Fischer Scientific, Inc., USA). The resulting peptide mixture wasanalyzed by LC–MS/MS on the LTQ-ORBITRAP mass spectrometer(Thermo Fischer Scientific, Inc., USA) coupled with 2D capillarychromatography Rheos 2000 (Flux instruments). The first dimen-sion column was Symmetry C18, 75 lm � 150 mm � 3 lm (LCPackings). Data dependent scan composed of one full MS scan (res-olution 60 000) and three CID MS/MS scans (resolution 7500) wasused as mass-spectrometry method. The mass tolerance of peptideidentification was 10 ppm. The mass tolerance for searching frag-ment ions was 50 ppm. The sequences have been searched in Uni-prot database by the Bioworks Browser 3.3.1 SP1 and sequest 2.0software (Thermo Fischer Scientific, Inc., USA).

2.3.2. N-terminal sequencingSapp1p was separated by 12% SDS–PAGE and transferred to

PVDF membrane using electro blotting apparatus (Trans-Blot SD,Bio-Rad). After Coomassie Blue staining, the appropriate bandwas cut and its N-terminal sequence was determined by Edmandegradation using an Applied Biosystems Procise sequencer.

2.3.3. Analytical size-exclusion chromatographySize-exclusion chromatography was performed on Superdex 200

10/300 GL Tricorn column (Pharmacia), equilibrated in 100 mMTris–HCl buffer at pH 7.0. Protein sample of 50 ll at 8 mg/ml wasanalyzed and the molecular weight was estimated by comparisonwith protein standards (Sigma–Aldrich) containing Blue dextran(2000 kDa), Bovine serum albumin (66 kDa), Carbonic anhydrase(29 kDa), Cytochrome C (12.4 kDa), and Aprotinin (6.5 kDa).

2.4. Proteolytic activity assays

Sapp1p proteolytic activity was determined by cleavage of thefluorogenic substrate Dabcyl-Glu-His-Val-Lys-Leu-Val-Glu-EDANS.Sapp1p cleaves the Leu-Val bond. The assays were performed in150 mM sodium citrate buffer, pH 3.75, at 37 �C for 2 h. The150 ll reaction mixture typically contained 5 ll of the substratestock solution (5 mg/ml in dimethyl sulfoxide) and 20 ll of the en-zyme solution (typical protein concentration was approximately0.05 mg/ml). The reaction was stopped by the addition of 20 ll of20% (v/v) trifluoroacetic acid, and the mixture was analyzed usingHPLC as previously described (Merkerova et al., 2006).

2.5. Crystallization and data collection

The Sapp1p–pepstatin A complex was prepared by mixing theenzyme with fivefold molar excess of pepstatin A (dissolved in di-methyl sulfoxide) and concentrated by ultrafiltration to concentra-tion of 8 mg/ml using Amicon Ultra-30 (Millipore). Crystals wereprepared at 19 �C using the hanging drop vapour diffusion tech-nique. The crystallization drop contained 2 ll of protein–pepstatinA complex solution, 1 ll of reservoir solution and 0.2 ll of 10 mMzinc acetate. The reservoir solution for the Sapp1p–pepstatin Acomplex contained 0.1 M Tris-Cl, pH 7.0, 2.0 M ammonium sulfateand 10% (v/v) glycerol. Micro-seeding technique was used for crys-tal optimization.

For data collection, the crystal was flash frozen in liquid nitro-gen. Diffraction data were collected at 100 K at beamline 19-BMof the Structural Biology Center at the Advanced Photon Source, Ar-gonne National Laboratory, Argonne, IL, USA. Diffraction data wereprocessed using the HKL-2000 suite of programs (Minor et al.,2006). Crystals exhibited the symmetry of space group P21 andcontained eight molecules in the asymmetric unit. Crystal param-eters and data collection statistics are given in Table 1.

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2.6. Structure determination and refinement

The phase problem was solved by molecular replacement usingprogram Molrep (Vagin and Teplyakov, 2000). The search modelwas derived from the structure of Sap2–A70450 inhibitor complexstructure (PDB code 1eag Cutfield et al., 1995). Molrep program ina ‘slow mode’ was used with three cycles of rigid body refinementbefore translation function (TF) and three cycles of rigid bodyrefinement after TF using diffraction data in resolution range44.3–4.0 Å. By this approach, seven molecules in the asymmetricunit (AU) were found with the R factor 43%.

Model refinement was carried out using the program REFMAC5.3 (Murshudov et al., 1997) from the CCP4 package (CCP4,1994). At the early stage of refinement, the electron density mapfor the eighth molecule in the AU was noticeable and the majorityof the protein chain was built automatically using program ARP/wARP (Perrakis et al., 1999). Manual protein and inhibitor buildingand adjustments were done using Coot (Emsley and Cowtan,2004). Tight non-crystallographic symmetry (NCS) restraints wereapplied during initial refinement, and in later stages, NCS restraintswere loosened as guided by the behavior of Rfree. No NCS restraintswere applied to the final refinement steps. The final refinementsteps included TLS refinement (Winn et al., 2001). The quality ofthe final model was validated with Molprobity (Lovell et al.,2003). Refinement statistics for all structures is given in Table 1.All figures showing structural representations were prepared withthe program PyMOL (DeLano, 2002). For structural analysis andcomparisons of two binding modes of inhibitor, chains A and Ewere used. The following services were used to analyze the struc-tures: PISA server (Krissinel and Henrick, 2005), Protein–proteininteraction server (Jones and Thornton, 1996), and STRIDE (Frish-man and Argos, 1995).

Atomic coordinates and experimental structure factors havebeen deposited in the Protein Data Bank with the accession code3FV3.

3. Results and discussion

3.1. Structure description and quality

The crystal structure of the Sapp1p from C. parapsilosis in com-plex with pepstatin A, a classical aspartic protease inhibitor, wasdetermined by molecular replacement using the structure ofSap2–A70450 inhibitor complex (Cutfield et al., 1995) as a searchmodel and refined using data to 1.85 Å resolution. To our knowl-edge, this structure represents the first crystal structure of C. par-apsilosis protein produced from its natural source. Themonoclinic crystal form contains eight protein–inhibitor com-plexes in the asymmetric unit with a solvent content of 49.8%.The final crystallographic model consists of eight molecules ofSapp1p–pepA complexes, each molecule containing 339 proteinresidues and one pepstatin A (PepA) molecule. All protein residuescould be modeled into a well-defined electron density map. Theroot mean square deviations (RMSDs) for superposition of the eightSapp1p protein molecules onto each other range from 0.178 to0.385 Å for the main-chain atoms, a value within the range ob-served for different crystal structures of identical proteins (Bettsand Sternberg, 1999). Minor structural changes are localized inthe surface exposed loops (residues 130–136, 247–251, and 287–292) and the substrate binding sites are structurally almost identi-cal. The electron density used for modeling of PepA was of excel-lent quality in all protein chains in the asymmetric unit.

Based on the electron density map interpretation, we foundthree residues in our Sapp1p model that differ with respect tothe original sequence published in the NCBI database (AccessionNo. P32951, de Viragh et al., 1993). The changes in positionLeu193 ? Ser, Pro196 ? Ala and deletion of Tyr between residues312 and 313 were confirmed by DNA sequencing and deposited toNCBI with the accession number FJ560879. The amino acid changesin the protein were further confirmed also by mass-spectrometryanalysis (data not shown). The exchange of Leu193 is due toambiguous codon usage in Candida species, where codon CUG isfrequently translated into serine (Miranda et al., 2006; Ohamaet al., 1993). Interestingly, mutation analogous to change ofPro196 into Ala in Sapp1p was described recently in a subunit of

Table 1Crystal parameters and diffraction data collection and refinement statistics.

Data collection statisticsSpace group P21

Cell parameters (Å) 86.5, 194.2, 97.2, 90.0, 91.5, 90.0Number of molecules in AU 8Wavelength (Å) 0.979Resolution (Å) 50.0–1.85 (1.9–1.85)Number of unique reflections 273 011Redundancy 3.8 (3.8)Completeness (%) 99.99 (99.94)Rmerge

a 4.5 (49.0)Average I/r(I) 27.2 (2.4)Wilson B (Å2) 24.4

Refinement statisticsResolution range (Å) 46.03–1.9 (1.9–1.85)No. of reflections in working set 269 660No. of reflections in test set 1368R valueb (%) 16.6Rfree valuec (%) 19.0RMSD bond length (Å) 0.012RMSD angle (�) 1.256Number of atoms in AU 23 536Number of protein atoms in AU 20 809Number of water molecules in AU 2150Mean B value (Å2) 15.1Ramachandran plot statistics

Residues in favored regions (%) 91.6Residues in allowed regions (%) 8.4

The data in parentheses refer to the highest-resolution shell.a Rmerge = (|Ihkl � hIi|)/Ihkl, where the average intensity hIi is taken over all sym-

metry equivalent measurements and Ihkl is the measured intensity for any givenreflection.

b R value = ||Fo| � |Fc||/|Fo|, where Fo and Fc are the observed and calculatedstructure factors, respectively.

c Rfree is equivalent to R value but is calculated for 0.5% of the reflections chosenin thin-resolution shells and omitted from the refinement process.

Fig. 1. Overall three-dimensional structure and secondary structure elements ofSapp1p complexed to pepstatin A. Protein in ribbon representation is colored bysecondary structure, pepstatin A molecule is represented by sticks.

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b-1,3-glucan synthase Fks1p of C. parapsilosis. This naturally occur-ring Pro ? Ala change in Fks1p is associated with a reduced sus-ceptibility of C. parapsilosis towards the echinocandin-classantifungals in comparison with C. albicans (Garcia-Effron et al.,2008).

The asymmetric unit of the Sapp1p crystal structure containseight protein–inhibitor complex molecules arranged into two tet-ramers related by non-crystallographic symmetry. The interfacearea of the tetrameric assembly is 5928 Å2, representing 11.5% ofthe overall accessible surface area of the four interacting mono-mers. Analysis of free energy barrier of tetramer dissociation(DGdiss) with PISA server (Krissinel and Henrick, 2005) yieldedDGdiss value of �25.1 kcal/mol. This negative value suggests thatthe stability of Sapp1p oligomeric assembly in solution is limited.Also, the size-exclusion chromatography revealed that Sapp1p ex-ists in solution exclusively as a monomer, even at high protein con-centrations used for crystallization (data not shown). Thus, the

tetrameric assembly of Sapp1p seems to be a crystallization arti-fact and the biologically relevant unit of Sapp1p is a monomer.

3.2. Overall fold of Sapp1p

Overall fold and topology of Sapp1p is very similar to the arche-typic fold of the monomeric aspartic protease family (Fig. 1). It ismainly a b-structure in which two domains form a central bindingcleft that accommodates a substrate. Each domain provides onecatalytic aspartate, which is part of the DTG or DSG motif. The cen-tral part of the extended active site is covered by anti-parallel b-sheet flap, a common feature of aspartic proteases which is inSapp1p formed by residues 71–89. The substrate binding site isin our structure occupied by PepA inhibitor, thus the flap adopts‘closed’ conformation of the active site.

The entrance to the funnel-like substrate binding site, includingthe non-primed substrate pockets, is lined by three so called

Fig. 2. Comparison of Sapp1p with orthologous enzymes from C. tropicalis (Sapt1p) and C. albicans (Sap2). (A) Sequence alignment of Sapp1p, Sapt1p, and Sap2. Secondarystructure elements found in Sapp1p are indicated. The Asp-Thr-Gly signature sequence is shown in bold, flap residues are indicated in italic and the changes of Leu193 ? Ser,Pro196 ? Ala and deletion of Tyr between residues 312 and 313 in Sapp1p are framed. Color shades indicate position of loops lining the substrate binding site: N-terminalentrance loop—yellow, N-terminal loop extended in Sapp1p—red, C-terminal loops 1, and 2—blue and green, respectively. (B and C) Top view into the active sites of Sapp1p–pepA (our structure), Sapt1p-peptide (PDB code 1j71) and Sap2–A70450 (PDB code 1eag). In (B) the enzymes are represented by their solvent accessible surface with loopscolored as in (A). Inhibitors and a peptide are shown as magenta sticks. In (C), the enzymes are represented by their solvent accessible surface colored by electrostaticpotential (red for negative, blue for positive).

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entrance loops, found also in other structures of Sap isoenzymes(Abad-Zapatero et al., 1996). The N-terminal entrance loop (resi-dues 47–53) is flanking the flap and two C-terminal entranceloops: C-ent loop 1 (residues 243–256) and C-ent loop 2 (residues285–292) are located across the binding cleft facing the flap and N-ent loop, respectively (Fig. 1).

The primed substrate pockets side of the active site is lined byan additional loop belonging to the N-terminal domain (residues126–139). This loop is formed by eight amino acid residues inser-tion and is not present in Sap isoenzymes from other Candidaspecies.

3.3. Comparison of substrate binding site with related structures

We compared the structure of Sapp1p with structures of twoorthologous isoenzymes from different Candida species: Sap2 fromC. albicans (Cutfield et al., 1995) and Sapt1p from C. tropicalis(Symersky et al., 1997). These isoenzymes are the most analogousin their physiological function and expression pattern in Candidalife cycle, since their secretion is induced by the presence of pro-teins as a sole nitrogen source (Hruskova-Heidingsfeldova, 2008).The sequence alignment suggests the sequence identity of Sapp1pwith Sapt1p and Sap2 is 57.5% and 55.5%, respectively (Fig. 2A).

The RMSD for superposition of Sapp1p with Sapt1p and Sap2structures is 0.94 Å for 332 aligned residues and 0.95 Å for 315aligned residues, respectively. The overall structure of Sapp1p isthus very similar to structures of Sapt1p and Sap2 with much ofthe secondary structure being conserved. However, there arenoticeable differences in structure of loops surrounding enzymeactive site. Although these loops are not in direct contact withinhibitor in Sapp1p structure, their conformation significantly af-fects the character, shape, and size of the substrate binding site.

The N-terminal entrance (N-ent) loop enclosed by two Cys res-idues forming disulfide bridge has been considered a characteristicfeature of aspartic proteases secreted by Candida species (Abad-Zapatero et al., 1996; Symersky et al., 1997). This loop in Sapt1pand Sap2 covers the non-primed substrate binding pockets alongwith the classical flap (Fig. 2). In Sapp1p this region is substantiallyshortened. While Sap2 and Sapt1p contain 11 amino acid residuesinserted between the bridged cysteines, in Sapp1p this insertion isonly five amino acid residues between Cys47 and Cys53. Thus res-idues 48–52 in Sapp1p form a short loop which does not reach overthe substrate binding and makes the S4 and S3 pockets more spa-cious and open.

Also the C-entrance loop 2 (C-ent 2) in Sapp1p formed by resi-dues 285–292 is much shorter compared to Sap2. While in Sap2,the C-ent 2 loop comprises 14 residues forming a long flexible loop,partially disordered in Sap2 crystal structure, this region in Sapp1pcomprises only seven residues folding into a short turn in-betweentwo anti-parallel b-strands. This resembles more of the size andtopology of this loop in Sapt1p (containing eight residues). Dueto deletions in both the N-ent loop and the C-ent 2 loop the Sapp1psubstrate binding cleft is thus widely open on its non-primed side.

On the other hand, the C-terminal loop 1 (C-ent 1) in Sapp1p(residues 243–256) contains an insertion of 4 or 1 residue com-pared to Sapt1p and Sap2, respectively. Dissimilar from the struc-ture of Sapt1p and Sap2, the C-ent loop 1 of Sapp1p forms anti-parallel b-sheets S16 and S17 (Fig. 1) positioned opposite to theflap. This loop conformation however can be a result of crystalpacking interactions. C-ent 1 loop is involved in crystal contactsand forms a four-stranded anti-parallel b-sheet with the same re-gion of the neighboring molecule.

An evident structural difference of the primed substrate pock-ets’ side of the active site in Sapp1p is a presence of an extendedloop belonging to the N-terminal domain (residues 126–139).This loop (designated here N-terminal loop 2) is formed by eight

amino acid residue insertion and is not present in Sap isoen-zymes from other Candida species nor in pepsin. N-terminal loop2 protrudes into the solvent region without any well-definedsecondary structure and partially covers the primed substratebinding pockets.

In comparison to Sapt1p and Sap2, the substrate binding site ofSapp1p is large and open on the non-primed side of the substratewhile the primed substrate pockets are shielded with N-terminalloop insertion. Analysis of electrostatic surfaces revealed that thebinding cleft of Sapp1p is much more negatively charged comparedto Sapt1p and Sap2. The strong negative patch in Sapp1p stretcheson both the primed and non-primed side of the active site (Fig. 2C).The differential size, shape, and character of the active site resultsin divergent substrate specificities and inhibitor affinities observedfor Sapp1p compared to Sapt1p and Sap2 (Majer et al., 2006). Forexample, Sapp1p is more sensitive to inhibition by the archetypalaspartic protease inhibitor pepstatin A (PepA). Sapp1p is inhibitedby PepA, with an inhibition constant (Ki) of 0.3 nM, while the Ki

values for Sapt1p and Sap2 are 5.1 and 1.4 nM, respectively (Pich-ova et al., 2001).

3.4. Pepstatin binding

PepA is an extended hexapeptide whose central statine residueforming a non-scissile bond, acts as a tetrahedral transition-statemimic. The PepA molecule was modeled into well-defined contin-uous electron density map present in the substrate binding grooveof all eight molecules of Sapp1 in the asymmetric unit. The high-quality of the map allowed us to distinguish between two alterna-tive conformations of PepA bound to various protein chains in theasymmetric unit (Fig. 3A). The two conformations (denominated

Fig. 3. Pepstatin A binding to Sapp1p. (A) The 2Fo-Fc electron density maps for twoalternate pepstatin A conformations is contoured at 1.0r. Conformations I and IIshown in magenta and green, respectively, and two catalytic aspartates aredepicted in sticks with carbon atoms colored yellow. (B) Superposition of PepAwhen bound to Sapp1p (magenta and green), Sap3 (yellow), and Sap5 (gray).

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conformation I and II) differ in the position of the C-terminal partof PepA, namely STA atoms CM, C, O and OXT (Fig. 3). Out of eightmolecules of the Sapp1p–PepA complex, conformation I was foundin five protein chains (A, B, C, D, and F) while conformation II waspresent in three protein chains (E, G, and H). The quality of theelectron density map was somewhat better for conformation IIcompared to conformation I. In conformation I, the C-terminalcarboxyl group is more exposed to the solvent and thus moredisordered due to higher flexibility and also due to partial decar-boxylation caused by radiation damage. Nevertheless, the qualityof the map was sufficient to unambiguously model the conforma-tion I (Fig. 3A).

PepA binds to Sapp1p in an extended conformation occupyingS4–S30 substrate binding subsites. The hydroxyl group of the cen-tral statine moiety forms two hydrogen bonds with the side-chainsof the two catalytic aspartates (Asp32 and Asp220). Further hydro-gen bonds involve interaction with main-chain atoms of Gly34 (O),

Gly79 (O and N), Asp80 (N), Gly222 (O), and Thr224 (N), and alsoside-chain atoms of Asp80 (OD2), Asn125 (ND2), Thr223 (OG1),and Thr224 (OG1). In addition to this hydrogen bonding network,there are extensive van der Waals interactions involving proteinresidues listed in Table 2. Upon the complex formation, the PepAburies 69.3% and 72.5% of its solvent accessible surface area forconformation I and II, respectively.

The PepA binding to Sapp1p can be compared with the threeavailable pepstatin-bound structures of Sap isoenzymes from C.albicans: Sap2 (Cutfield et al., 1995), Sap3 (Borelli et al., 2007),and Sap5 (Borelli et al., 2008). PepA binds to Sapp1p in a very sim-ilar conformation as observed for structures of Sap3 (PDB code2h6t) and Sap5 (PDB code 2qzx). Residues involved in PepA bind-ing as well as the hydrogen bonding pattern for the central bindingpockets (S3, S2, S1, and S10) are mostly conserved in analyzed iso-enzymes (Table 2). Differences are observed in subsites S4, S20, andmostly S30. The major difference in PepA binding and conformation

Table 2Residues in contact (<4 Å) with PepA in Sapp1p structure known.

Subsite Sapp1p Sap2 Sap3 Sap5

S4 12 Val 12 Val 12 Ile224 Thr 222 Thr 222 Thr 222 Thr225 Leu 223 Ile227 Tyr 225 Tyr

51 Trp83 Lys84 Tyr85 Gly

285 Tyr

S3 12 Pro 12 Val 12 Val 12 Ile80 Asp 86 Asp 86 Asp 86 Asp

222 Gly 220 Gly 220 Gly223 Thr 221 Thr 221 Thr 221 Thr224 Thr 222 Thr 222 Thr 222 Thr

S2 78 Tyr 84 Tyr 84 Tyr 84 Tyr79 Gly 85 Gly 85 Gly 85 Gly80 Asp 86 Asp 86 Asp 86 Asp

220 Gly 220 Gly 220 Gly223 Thr 221 Thr 221 Thr227 Tyr 225 Tyr 225 Tyr 225 Tyr

83 Lys

S1 303 Ile 305 Ile30 Ile 30 Ile 30 Val32 Asp 32 Asp 32 Asp 32 Asp34 Gly 34 Gly 34 Gly 34 Gly78 Tyr 84 Tyr 84 Tyr 84 Tyr79 Gly 85 Gly 85 Gly 85 Gly80 Asp 86 Asp 86 Asp82 Ser

117 Ile 123 Ile 123 Ile 123 Ile

S10 34 Gly 34 Gly 34 Gly 34 Gly35 Ser 35 Ser 35 Ser 35 Ser

125 Asn220 Asp 218 Asp 218 Asp 218 Asp222 Gly 220 Gly 220 Gly 220 Gly223 Thr 221 Thr 221 Thr

S20 82 Ile79 Glya 83 Gly 83 Glu 83 Lys

84 Tyr 84 Tyr 84 Tyr85 Gly

131 Gly133 Ala

218 Leua

301 Aspa

S30 79 Glyb

301 Aspb 303 Tyr195 Glnb 193 Glu

Residues forming hydrogen bonds with PepA are shown in bold.a Residues interacting with PepA conformation I.b Residues interacting with PepA conformation II.

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is found in the statine residue in P30 position (Fig. 3B). In Sapp1p,this residue is positioned much closer to the C-terminal domainportion of the substrate binding cleft and makes van der Waalscontacts with residues Asp301, Gln303, and Gln195. Differentialbinding of the PepA P30 residue might be caused by the presenceof the inserted N-terminal loop 2 which extends over the S30 sub-strate binding site of Sapp1p and changes its shape and charactercompared to other Sap enzymes.

In general, the S30 site does not play a crucial role in the sub-strate or inhibitor binding to Sap isoenzymes and aspartic prote-ases since the P30 residues usually make very few contacts withthe enzyme (Bailey and Cooper, 1994). Strikingly, in Sapp1p theP30 residue of PepA is located in the proximity of the region con-taining two substitutions identified in our structure: Leu193 ? Serpresent in Candida species due to ambiguous codon usage andmutation Pro196 ? Ala. Residues 193 and 196 are located withina short loop between S12 and S13 b-strands whose tip residue(Gln195) is in direct interaction with P30 of PepA conformation II(Fig. 4).

The reassignment of the CUG codon to serine in Candida spp.was studied from the point of view of the genetic code evolution(reviewed in Miranda et al., 2006) nevertheless, the impact of theLeu ? Ser change on structure and function of pathogenic Candidaspp. proteins has only been addressed scarcely. Cutfield et al.(2000) examined structures of exo-b-1,3-glucanase from C. albi-cans, containing either leucine or serine in the position correspond-ing to the only CUG codon in the enzyme sequence. This position issituated in a helix on the surface of the protein, distant from theactive site, and the residue side-chain points out from the proteinsurface. Thus, the Leu ? Ser change has only a minimal effect onthe structure and function of the exoglucanase.

The proximity of the Leu ? Ser change to the P30 residue ofPepA in our Sapp1p structure encouraged us to revisit the resultsof our previous work (Dostal et al., 2005), which described pro-cessing of a recombinant Sapp1p precursor. This precursor wasexpressed in Escherichia coli, contained Leu at the position 193,and was capable of autocatalytic activation, although the cleav-age occurred one amino acid downstream from the expectedpromature junction. The resulting protein (designated Sapp1p�1)displayed similar kinetic properties as the authentic Sapp1p iso-lated from the C. parapsilosis culture medium. The kinetic com-

parison was performed using three oligopeptide substrates andseven active site targeted inhibitors. The Ki values for pepstatinand KM and kcat values for the most frequently used substrateLysProAlaGluPheNphAlaLeu were essentially the same both forauthentic Sapp1p and for Sapp1p�1, showing that neither trunca-tion by the N-terminal amino acid, nor the Leu ? Ser change atthe position 193 have a significant impact on the function ofSapp1p. Nevertheless, one substrate and three inhibitors showedslightly worse kinetic constants for Sapp1p�1. Although thesedifferences did not significantly exceed the standard deviation,they indicate that attention should be paid to subtle variations,which might result from the ambiguous usage of CUG codon.More importantly, the properties of loop 192–197 may be sub-stantially different in the case of simultaneous presence ofLeu193 and Pro196. The Sapp1p species containing both of thesemutations will be further characterized in order to elucidatewhether the naturally occurring amino acid changes might playa role in regulation of extracellular proteolytic activity of C. par-apsilosis during the evolution.

4. Conclusion

We have solved the first structure of Sap isoenzyme from Can-dida parapsilosis isolated from its’ natural source in complex withpepstatin A. The overall secondary structure of Sapp1p resemblesknown structures of other Sap isoenzymes from C. albicans and C.tropicalis, however, striking alterations in the structure of theloops lining the substrate binding pocket were observed. Due todeletions in both the N-terminal entrance loop and the C-terminalentrance loop 2 the Sapp1p substrate binding cleft widely openson its non-primed side. The primed side of the substrate bindingcleft is more closed due to eight amino acid residue insertion(residues 126–139) forming a loop which covers the S30 substratebinding pocket. The unique shape and character of the active siteexplain divergent substrate specificities observed for Sapp1pcompared to Sapt1p and Sap2 and result in differential bindingof P20 and P30 residues of pepstatin A. Detailed analysis of pro-tein–inhibitor interactions also revealed interaction of P30 residuewith the loop containing the substitution Leu193 ? Ser due toambiguous codon usage in Candida species and mutationPro196 ? Ala.

Sapp1p plays an essential role in the virulence of C. parapsilosis,one of the most common causes of Candida infections. The enzymefacilitates the pathogen invasion of the host tissues and enablesmicroorganisms to utilize host cell macromolecules as a nutrientsource. Although the panels of the active site peptidomimeticinhibitors synthesized to date have not resulted in a design ofdrugs suitable for treatment of systemic infections, the compoundsdirected against Sap isoenzymes might be useful as antimycoticsfor topical administration. Structural studies aimed at Sap-inhibi-tor complexes contribute to understanding of the class of enzymesthat act as important virulence factors of various pathogenic Can-dida species and aid in structure-based design of Sap-specificinhibitors.

Acknowledgments

The authors thank Miloslav Šanda for performing the peptidemass fingerprinting analysis, Klára Grantz Šašková for help withprotein crystallization, Zdenek Voburka for N-terminal proteinsequencing.

This work was supported by the Ministry of Education of theCzech Republic (Research Program No. LC531), and research pro-jects AV0Z40550506, and AV0Z50520514 awarded by the Acad-emy of Sciences of the Czech Republic.

Fig. 4. Detail of pepstatin A interaction with loop containing Leu193 ? Ser changedue to ambiguous codon usage and mutation Pro196 ? Ala. Pepstatin A inconformation II and loop 192–197 are represented by sticks and their solventaccessible surfaces are colored violet and yellow, respectively. Hydrogen bondbetween Gln195 and PepA is shown as a green dashed line, the number representsthe distance in Å. The red line represents N-terminal loop 2 insertion.

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