Subtilisin Inhibitor and the Subtilisin Savinase Reveal a Novel Mode of Inhibition

doi:10.1016/j.jmb.2008.05.034

J. Mol. Biol. (2008) 380, 681–690

Available online at www.sciencedirect.com

Structural and Mutational Analyses of the Interaction between the Barley α-Amylase/Subtilisin Inhibitor and the Subtilisin Savinase Reveal a Novel Mode of Inhibition Pernille Ollendorff Micheelsen 1 ⁎, Jitka Vévodová 2 , Leonardo De Maria 1 , Peter Rahbek Østergaard 1 , Esben Peter Friis 1 , Keith Wilson 3 and Michael Skjøt 1 1

Research and Development, Novozymes A/S, Krogshøjvej 36, 2880 Bagsværd, Denmark 2

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK 3

York Structural Biology Laboratory, University of York, Heslington, York YO10 5YW, UK

Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley α-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone. © 2008 Elsevier Ltd. All rights reserved.

Received 5 February 2008; received in revised form 9 May 2008; accepted 14 May 2008 Available online 22 May 2008 Edited by R. Huber

Keywords: barley α-amylase/subtilisin inhibitor; subtilisin; Savinase; inhibition; X-ray crystallography

Introduction Subtilisins represent a large class of microbial serine proteases, of which those secreted by the Bacillus species (e.g., BPN′ from Bacillus amyloliquefaciens, subtilisin Carlsberg from Bacillus subtilis and Savinase from Bacillus lentus) are the most studied. Subtilisins are endopeptidases with a catalytic triad consisting of Asp32, His64 and Ser221 (with BPN′ numbering used consistently1). Subtilisins are globular proteins, with the active site situated in a shallow groove on the surface. The mechanism is *Corresponding author. E-mail address: [email protected]. Abbreviations used: BASI, barley α-amylase/subtilisin inhibitor; WASI, wheat α-amylase/subtilisin inhibitor; CI-2, chymotrypsin inhibitor 2; AMY2, α-amylase 2; SAS, solvent-accessible surface.

identical with that of trypsin-like family enzymes but is evolutionary and structurally different.2 The substrate binding cleft of Savinase provides six subsites for binding of the side chains of the protein to be degraded, designated S4–S2′ (nomenclature according to Ref. 3). Inhibitor residues corresponding to the subsites are numbered P4–P2′, and adjacent inhibitor residues are numbered sequentially; cleavage occurs between the P1 and P1′ residues. More than 50% of the 275 amino acids have been subjected to mutagenesis, and this has provided great insight into enzyme catalysis.4 Furthermore, subtilisins are important industrial enzymes, being used in various fields such as detergent production, food production, feed production, leather and textile processing, pharmaceutical production, diagnostics and waste management.5 The three-dimensional fold is highly conserved, with a central seven-stranded parallel β-sheet flanked on both sides by α-helices.6–8 There is a highly conserved calcium binding site in most Bacillus sub-

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682 tilisins coordinated by an aspartic acid and five carbonyl oxygens, four of which are found in the loop made up by residues 75–81. There is a second and much weaker ion binding site on the surface 32 Å away from the strong calcium site that coordinates to residues 195 and 197. This region actually has two ion binding sites close to each other; one accommodates sodium and the other accommodates calcium. These two sites cannot be occupied simultaneously.4 Complexes of the serine proteases with proteinaceous inhibitors have been the object of intensive study. Inhibitors identified to date fall into about 20 structural families.9 Proteinaceous subtilisin inhibitors are found in 3 families [Potato I inhibitor family, Streptomyces subtilisin inhibitor (SSI) family and Kazal inhibitor family7,10,11] and are all canonical inhibitors. The structures of inhibitors from all 3 classes have been solved in complex with a subtilisin: chymotrypsin inhibitor 2 (CI-2, from the Potato I inhibitor family)7 and SSI (from the SSI family) both in complex with subtilisin Novo (BPN′)10 and the turkey ovomucoid third domain (OMTKY3, from the Kazal inhibitor family) in complex with subtilisin Carlsberg.11 Although the 3 families of inhibitors have different overall folds, they all interact with subtilisin via a structurally conserved exposed loop surrounding the fully exposed P1 residue. The interaction is stabilised through a highly conserved hydrogen bonding pattern.7,12–15 Hordeum vulgare α-amylase/subtilisin inhibitor (BASI) is a double-headed inhibitor that acts both on α-amylase 2 (AMY2) from barley and on serine proteases from the subtilisin family. BASI is thought to have two functions: to control degradation of starch during premature sprouting16 and to protect the seed from pathogen-derived subtilisin-type serine proteases17 (for a review, see the work of Bellincampi et al.18 and Nielsen et al.19). It is a single-chain protein of 181 amino acids and belongs to the Kunitz-P-type inhibitor family. 16,20,21 BASI shares about 30% sequence identity with Kunitz-type inhibitors from other plants that contain two conserved disulfide bridges.22 The closest homologues to BASI are from wheat (WASI) and rice, and these share 92% and 58% sequence identities, respectively.23,24 The structure of BASI has been solved in complex with AMY225 and barley thioredoxin (HvTrxh2)26: In both complexes, BASI displays the β-trefoil topology. BASI does not inhibit barley thioredoxin but rather serves as a substrate. In this study, we present the X-ray structure of BASI in complex with the B. lentus subtilisin Savinase, which shows that BASI inhibits subtilisins in a novel way.

Results and Discussion Architecture of the BASI–Savinase complex There are two independent complexes in the asymmetric unit, with very similar structures: superpo-

Novel Inhibition Mode of a Subtilisin

sition of the two results in a root-mean-square (rms) difference of 0.8 Å over 450 Cα atoms. The inhibitor binds to the active site of the enzyme, situated in a shallow groove on the surface (Fig. 1), in agreement with biochemical experiments that supported a strictly competitive inhibition mode.27 Eighteen residues of the inhibitor interact with 28 residues of the enzyme. The overall contact area between the enzyme and the inhibitor is 1800 Å2, which is equivalent or slightly higher than areas in other subtilisin–proteinaceous inhibitor complexes (Fig. 2a, upper panel).28 In the standard mechanism for canonical protein inhibition of serine proteases, the inhibitor binds to the protease in a substrate-like manner, with the protease interacting loop kept in a well-ordered conformation. BASI residues Ala86– Thr89 bind to Savinase S5 to S2 by hydrogen bonding. At the interface, there are 144 contacts less than 4 Å. BASI residues Ala86–Thr89 (P5–P2) make 77 contacts with 17 residues in Savinase, 7 of which are hydrogen bonds. In the cleft leading to the active site, the P4–P2 residues make five main chain–main chain hydrogen bonds, which follow the expected hydrogen bonding pattern typical for P4–P2 interactions (Fig. 2, lower panel).6 Two additional side chain– main chain hydrogen bonds are formed between BASI Ala86–Savinase Ser126 and BASI Tyr87–Savinase Ser130. Savinase fold and mobility Savinase displays the subtilisin-like fold with the same topology as reported previously.2 Superposition of the Cα atoms from the two Savinase molecules in the asymmetric unit gives an rms difference of 0.3 Å for 269 Cα atoms. Superposing the current model on native Savinase (1SVN)2 gives a similar rms of 0.3 Å over the same 269 Cα atoms. There is no significant rearrangement of Savinase side chains in the active site on BASI inhibition. BASI fold and mobility In the complex, BASI displays the β-trefoil topology with the same topology as reported previously.25,26 Superposition of the 181 Cα atoms from the two independent BASI molecules gives an rms difference of 1.1 Å. Furthermore, BASI can be superposed with the BASI molecule (chain C) in complex with AMY2 (1AVA)25 and with the BASI molecule in complex with HvTrxh2 (2IWT),26 resulting in rms differences of 2.3 and 1.8 Å, respectively (based on 181 and 179 Cα atoms, respectively). The relatively high differences for superposition of BASI are due to the high level of flexibility of the loops (definition of loops and β-strands as in Vallee et al.25). Twenty-eight residues, all situated in loops found on the surface of BASI, have B-factors above 50. When those residues are omitted from the superposition and the remaining 153 Cα atoms are superposed, the rms difference drops to 0.8 Å, whereas if only the core structure, omitting all loops, is superposed, the rms is 0.5 Å (62 Cα). In the BASI–AMY2 complex, a large part of

683

Novel Inhibition Mode of a Subtilisin

Fig. 1. Overall structure of the BASI–Savinase complex. BASI is shown in red; Savinase, in blue; the calcium in site A, in green; and the sodium in site B, in yellow.

the BASI surface is tightly bound in the interface; the loops involved in the BASI–AMY2 interface are therefore in a different conformation compared with the BASI–Savinase complex. The rms value drops to 1.4 Å (113 Cα atoms used, BASI–Savinase chain C and 1AVA chain C) when residues involved in the AMY2 binding are omitted from the superposition (Fig. 3).25

Positioning of the metal ions All subtilisins are calcium dependent, and many of the Bacillus enzymes have a conserved strong calcium binding site (site A) and a weaker ion binding site (site B).2,7,8 In addition to the four peptide chains, 4 calcium ions, 6 sodium ions and 24

Fig. 2. Subtilisin inhibition modes. Upper panel: The subtilisin surface is shown in white, and residues in contact with the inhibitor are shown in blue. Residues of the inhibitor spanning subsites P4′ to P9 are shown as red sticks. Lower panel: Zoom-in on the interaction. P5–P2′ inhibitor residues and interacting subtilisin residues are shown as sticks; subtilisin residues, active site residues and inhibitor residues are shown in blue, yellow and red, respectively. β-Strands are shown as arrows. (a) BASI (this work, chains A and C). (b) SSI (2SIC). (c) CI-2 (2SNI). (d) OMTKY3 (1YU6, chains A and C).

684

Novel Inhibition Mode of a Subtilisin

Fig. 3. BASI plasticity I. Cα–Cα distance between corresponding BASI residues after superimposition onto chain C of the BASI–Savinase complex; loops are marked with gray shading. Upper panel: BASI (this work, chain D). Middle panel: AMY2–BASI complex (2AVA, chain C). Lower panel: HvTrxh2–BASI complex (2IWT). In the small panel at the bottom, orange, red and yellow squares identify BASI residues in contact with Savinase, AMY2 and HvTrxh2, respectively.

chloride ions were modelled into the electron density. All identified ions are present in the crystallisation solution. Two of the calcium ions are bound in site A and coordinate to Savinase as previously described.2,7,8,29 The other two calcium ions are coordinated to Savinase Val153 and four water molecules; there is no coordination with BASI. Site B is occupied by a sodium ion in both complexes (Fig. 1). Comparison of the two complexes in the asymmetric unit The overall structures of the two complexes are essentially identical. However, there are some small differences, with one significant difference in the enzyme–inhibitor interface. The buried surface area in the interface of the AC complex is 1866 Å2, while that of the BD complex is 1798 Å2.28 In the interface, only two BASI residues, Arg61 and Arg85, differ significantly in the interface area contribution, due to side chain rearrangement (Fig. 4). The solvent-accessible surface (SAS) for the two residues was calculated (Table 1) in order to better understand the effects of the side chain rearrangements on the interface area. The biggest difference arises from Arg61. Taking the entire residue into account, 77% and 86% of the SAS are covered by BASI itself in the C and D chains, respectively; in addition, when BASI is bound to Savinase, 70% and 0% of the SAS are covered by Savinase in the AC and BD complexes, respectively. Thus, Arg61 contributes to the interface area with 60 and 0 Å2 in the AC and BD complexes, respectively. The opposite situation is seen for Arg85,

which contributes to the interface area with 40 and 80 Å2 in the AC and BD complexes, respectively. Furthermore, in the AC complex, BASI Ala181 has seven contacts with Savinase Thr132, two of which are hydrogen bonds; on the other hand, in the DB complex, Ala181 is not involved in the interface. In general, there are more contacts and hydrogen bonds in the AC complex compared with the BD complex. These differences probably reflect differences in the crystal contacts of the two complexes. The D chain is more solvated than the C chain, and this could explain the difference observed in the mobility of the two chains. Comparison with other subtilisin–inhibitor complexes BASI interacts with Savinase via an exposed loop, but the length of the loop differs from that observed in the complexes previously reported (Fig. 2). Whereas the subtilisin interacting loops in the previous structures constitute the P4–P2′ residues, the loop in BASI is composed of the P5–P2 residues (Ala86– Thr89). After the P2 residue, the BASI loop is pulled out of the Savinase cleft, as Cys90 (which would be the P1 residue) forms a disulfide bond with Cys43. This moves the potential scissile bond, between Cys90 and Leu91, 6.4 Å away from the active serine, compared with about 2.6 Å in the other three types of inhibitors (Fig. 2, lower panel). Since the interacting loop in BASI is shorter, the triple-stranded β-sheet seen in other subtilisin–proteinaceous inhibitor complexes is missing (Fig. 2, lower panel). This is a novel

685

Novel Inhibition Mode of a Subtilisin

Fig. 4. BASI plasticity II. The side chains of the interacting residues are shown as sticks; BASI residues are shown in red, and the surface of Savinase is shown in blue. (a) BASI–Savinase, corresponding to chains A and C. (b) BASI–Savinase, corresponding to chains B and D of this work.

way for a proteinaceous inhibitor to interact with a subtilisin. The inhibition is basically canonical, but as the interacting loop is shorter, it lacks the reactive bond. As the potential scissile bond is pulled away from the active serine, BASI is not degraded, in contrast to other known serine protease inhibitors where the P1–P1′ bond is slowly degraded by the enzyme.30–32 This suggests that the BASI–subtilisin complex is more stable than other known subtilisin– inhibitor complexes. The cysteine that prevents BASI from being degraded is conserved in the close homologues in WASI23 and rice α-amylase/subtilisin inhibitor,24 which can be assumed to share this novel mode of interaction. Biological role and regulation of BASI In addition to inhibiting Savinase, BASI inhibits AMY2 from barley, and indeed both enzymes can be inhibited simultaneously.16,33 The Savinase inhibi-

Savinase inhibition by BASI mutants

Table 1. SASs for Arg61 and Arg85 (Å2) AC

tion is calcium independent, while the AMY2 inhibition is calcium dependent, as there is a fully hydrated calcium ion in the interface between BASI and AMY2 that is absent from the AMY2 structure.25 It has been suggested that the calcium ion in the BASI–AMY2 interface plays an important role in the regulation of the AMY2 inhibition.33 Amylases in grains must be inhibited to prevent premature sprouting, but the inhibition needs to be regulated in order for the seed to germinate at a later point. The ability of BASI to simultaneously inhibit AMY2 and Savinase, the possible regulatory role of the calcium ion in the BASI–AMY2 interaction and the unregulated nature of the BASI–Savinase interaction are consistent with the proposed biological roles of BASI: to prevent premature sprouting and to inhibit pathogenderived proteases. The AMY2 and the Savinase binding sites are located on opposite surfaces of BASI (Fig. 5). Therefore, the ternary complex between AMY2, BASI and Savinase might occur naturally, although it has not yet been isolated to date.

BD

ISO

BASI

CPLX

ISO

BASI

CPLX

Arg61 Side chain Backbone Total

233.00 151.78 384.78

84.59 0.42 85.02

24.63 0.42 25.05

228.72 143.53 372.25

48.23 0.51 48.74

48.23 0.51 48.74

Arg85 Side chain Backbone Total

228.01 145.87 373.88

46.13 4.47 50.60

10.83 0.00 10.83

229.21 145.10 374.31

87.34 9.83 97.17

16.53 0.12 16.65

The residues were considered as isolated (ISO), in the context of BASI (BASI) and in the context of the enzyme (CPLX). The contributions were split into backbone and side chain.

The binding loop of CI-2 is supported and conformationally constrained by an extensive network of hydrogen bonds and intramolecular interactions.7,34 This network is not present in BASI, but BASI has a more extended hydrogen bond network between the P5–P2 residues and the residues in the subtilisin groove. Although BASI and CI-2 hydrogen bond to Savinase in the same manner with respect to the P4– P2 residues and the cores of the loop consisting of the P7–P2 residues have the same conformation, only the P2 residue is conserved (BASI Thr89 and CI-2 Thr58). All BASI P7–P2 residues were mutated individually to those present in the CI-2 P7–P2 sequence to gain a better understanding of the role of each residue in the

686

Novel Inhibition Mode of a Subtilisin

Fig. 5. The ternary complex AMY2–BASI–Savinase. AMY2 is shown in yellow; BASI, in red; Savinase, in blue; and the calcium situated in the AMY2–BASI interface, in green.

inhibition. In addition, Y87A and T89A, two BASI point mutations, previously reported to have a 3.5fold decrease in affinity and no affinity change, respectively,35 were included in this study. The effect of the mutations on the inhibition of Savinase was assayed on N-Suc-AAPF-pNA (Nsuccinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide). The Ki values increased for all mutants, ranging from a few to more than 120-fold (Fig. 6 and Table 2). Mutation of the outermost residues of the P7–P2 loop (P7, F84V; P6, R86G; P2, T89A) had little effect on Ki, while mutation of the P3–P5 residues resulted in changes ranging from 11-fold to more than 120-fold. Both the A86T and the Y87T mutants showed a 25- to 30-fold reduction in Ki. Ala86 is situated in a hydrophobic pocket, and, presumably, introducing a hydrophilic residue destabilises the interaction. In the wild-type BASI, Tyr87 is involved in a side chain hydrogen bond to Savinase Ser130. When mutated into a residue with a shorter side chain, the hydrogen bond is presumably lost; the Y87A mutant showed an 11-fold decrease in affinity. The Y87A mutant was previously reported to increase Ki by only 3.5-fold35 compared with the 10fold increase seen here. In addition, Bønsager et al. reported a value for Ki for the wild-type enzyme higher than that measured in this study. The differences seen in the two studies must be due to experi-

mental differences, including assay conditions, protein expression, protein purification method and protein modifications. The most significant change in inhibition effect results from mutation of the P3 residue (T88V). The side chain of Thr88 forms two intramolecular hydro-

▪

Fig. 6. Inhibition of Savinase by BASI wild type and BASI variants. ( ) BASI wild type; (▴) F84V; (O) R85G; (●) A86T; (□) Y87I; (△) Y87A; (◊) T88V; (○) T89A. The inhibition assay was carried out at pH 9.8 (see Materials and Methods). Curves are drawn through the experimental points.

687

Novel Inhibition Mode of a Subtilisin Table 2. Ki values for Savinase inhibition by BASI variants Inhibitor

Ki/nM

Wild type F84V R85G A86T Y87I Y87A T88V T89A

4.5 (±0.6) 9.0 (±1.2) 8.2 (±1.1) 100.0 (±14.6) 91.1 (±12.4) 40.3 (±5.3) 541.8 (±250.4) 6.0 (±0.8)

All experiments were carried out in triplets.

gen bonds to Cys90 and Leu91; in addition, there are several intramolecular interactions between the Thr88 side chain and the BASI backbone, which are presumably lost when valine is introduced. From the present study, it is clear that Ala86, Thr87 and especially Thr88 are important for Savinase inhibition, and all are conserved in the wheat inhibitor, while only Ala86 and Thr88 are conserved in the rice inhibitor.23,24 The variation in loop sequence in subtilisin inhibitors suggests that the side chains play a major role in constraining the loops in the right conformation necessary for subtilisin inhibition. Sequence conservation of protease inhibiting loops Within inhibitor families, there is high sequence variability in the interacting loops, with some clear amino acid preferences. The Kunitz-P-type inhibitors from soybean have a conserved proline at the P3 position, whereas in the barley, wheat and rice inhibitors, there is a threonine.9 The present study shows that threonine is crucial for BASI inhibition of Savinase. In canonical inhibitors, the side chain of the P3 residue is not involved in the interface with the enzyme, as it points away from the enzyme and towards the solvent or the inhibitor itself.6 In contrast, in the BASI–Savinase complex, the side chain of Thr88 forms hydrogen bonds with the backbones of Cys90 and Leu91, leading to stabilisation of the P5– P2 loop. When the polar OH moiety of Thr88 is replaced by a hydrophobic CH3 (T88V), the loop is no longer restrained in the conformation necessary for binding, and the inhibitory power is lost. Proteinase K inhibition by cereal Kunitz-P-type inhibitors A low-resolution structure of the WASI, which shares 92% sequence identity with BASI, has been solved in complex with proteinase K.36 Given the high sequence identity between BASI and WASI, the ability of BASI to inhibit proteinase K was tested, and it was observed that BASI inhibits proteinase K to the same extent as it inhibits Savinase (data not shown). A model of the complex between BASI and proteinase K was generated by superposing proteinase K [Protein Data Bank (PDB) ID 1OYO] onto Savinase in complex with BASI (the coordinates for the pro-

teinase K–WASI complex are not available). Although proteinase K and Savinase share only 37% sequence identity, the region around the active site superposes well and the enzyme–inhibitor interface, including the hydrogen bonding pattern, is conserved. The only clash in the interface is seen between the side chain of proteinase K Tyr104 and BASI Tyr87. However, a significant side chain rearrangement has been reported in the proteinase K complex with WASI, leading to stacking of proteinase K Tyr104 and WASI Tyr86 (equivalent to BASI Tyr87), thereby contributing to the tight binding in this complex.36 It is reasonable to assume that the same side chain rearrangement would occur in a proteinase K complex with BASI.

Conclusions In this study, the first cereal Kunitz-P-type inhibitor in complex with a subtilisin has been determined. BASI inhibits subtilisin in a novel way, as the interacting loop only includes the P4–P2 residues, while the interacting loop of inhibitors from other families consists of the P4–P2′ residues. The interacting loop is pulled away from the active serine by a disulfide bridge between Cys90 and Cys43, preventing cleavage of the inhibitor. BASI also inhibits proteinase K, and it is reasonable to assume that the same residues are involved in the interface, based on superposition of Savinase and proteinase K. The roles of the loop residues have been analysed by mutating them one by one. All seven variants showed increased Ki values, ranging from a few times to more than 120 times. The P3 residue (Thr88) is shown to be crucial for the inhibition, as it stabilises the conformation of the interacting loop, through hydrogen bonding and intramolecular interactions with the BASI backbone.

Materials and Methods Cloning All constructs were cloned as described previously.37 The BASI gene was synthesised by PCR-based gene synthesis.38 All oligonucleotides were purchased from Invitrogen. BASI cDNA without signal sequence and the truncated α-factor secretion signal37 was amplified and combined using splicing-by-overlapping extension PCR. All constructs were subcloned in the TOPO vector using a TOPO Blunt TOPO PCR Cloning Kit (Invitrogen). Variants were generated by PCR methods essentially like QuickChange (Stratagene) using a BASI template. The following internal mutagenic primers were used to i n t r o d u c e m u t a t i o n s ( u n d e r l i n e d ) : F 8 4 V, 5 ′ GTACGCATATCCGTCCGCGCCTACACG-3′; R85G, 5′CGCATATCCTTCGGCGCCTACACGACG-3′; A86T, 5′CATATCCTTCCGCACCTACACGACGTGTC-3′; Y87I, 5′-CCTTCCGCGCCATCACGACGTGTCTG-3′; Y87A, 5′-CCTTCCGCGCCGCCACGACGTGTCTGC-3′; T88V, 5′-CTTCCGCGCCTACGTGACGTGTCTGCAG-3′; and T89A, 5′-CGCGCCTACACGGCCTGTCTGCAGTCC-3′.

688 Protein expression, purification and characterisation Wild-type BASI and all seven variants were expressed as reported previously.37 Mutation did not alter the expression levels, indicating proper global folding of the proteins. Molecular size and purity were verified by SDS-PAGE and mass spectroscopy (not shown). BASI was precipitated from the supernatant by adding solid ammonium sulfate [(NH4)2SO4] to a final concentration of 3.2 M, and the solution was stirred for 30 min. The precipitate was collected by centrifugation (20,000g, 20 min) and dissolved in a minimal volume of buffer A (20 mM CH3COOH/NaOH, pH 5.0). The dissolved BASI was transferred to buffer A by gel filtration (desalting) on a G25 Sephadex column. The desalted BASI solution was slightly turbid, and this turbidity was removed by 0.45-μm filtration. The filtrate was applied to a HighLoad SP-Sepharose column equilibrated in buffer A. After washing with buffer A, the column was eluted with a linear NaCl gradient (0 → 0.5 M) in buffer A over 10 column volumes. Fractions from the column with BASI were pooled and diluted 10 times with deionised water to reduce the conductivity. The diluted BASI pool was applied to a SOURCE S column equilibrated in buffer A. After washing with buffer A, the column was eluted with a linear NaCl gradient (0 → 0.5 M) in buffer A over 10 column volumes. BASI-containing fractions from the column were analysed by SDS-PAGE, and pure fractions were pooled. G25 Sephadex, HighLoad SP-Sepharose and SOURCE S are column materials from GE Healthcare. Crystallisation Savinase was purified by affinity chromatography and ion-exchange chromatography. BASI and Savinase were mixed with a small molar excess of BASI. The mixture was dialysed overnight in 10 mM Mes (4-morpholineethanesulfonic acid) and 2 mM CaCl2, pH 6.0, and concentrated by centrifugation in an Amicon Ultra-15 device (with a cutoff of 5000 Da, Millipore Corporation). The BASI–Savinase complex was concentrated to 23 mg ml− 1 in 10 mM Mes buffer, pH 6.0, and 2 mM CaCl2. Crystallisation screening was conducted using sitting-drop vapour diffusion with Crystal Screen 1, Crystal Screen 2, Index Screen (all from Hampton Research) and PACT Screen (Molecular Dimensions Limited). Small single crystals were obtained in index screen condition A9 (0.1 M Bis–Tris, pH 5.5, and 3 M NaCl). Subsequently, hanging-drop vapour diffusion was applied to optimise those conditions that had led to the most promising results, as well as exploring a range of different pH levels and types and concentrations of precipitant. Drops containing 1 μl of the protein and 1 μl of reservoir solution were equilibrated against 500 μl of reservoir solution at 18 °C. Crystals suitable for X-ray diffraction were obtained from 0.1 M sodium acetate buffer, pH 5.6, and 4 M NaCl in the form of bipyramids of average dimensions 0.4 mm × 0.3 mm × 0.3 mm, which appeared after 5–10 days. Data collection and processing Prior to data collection, crystals were mounted in nylon CryoLoops (Hampton Research) and vitrified. No cryoprotectant was required due to the high concentration of NaCl in the mother liquor. Data were collected at ESRF ID23-1 at 100 K with an ADSC Q315 detector. The crystals diffracted to a resolution of 1.85 Å and belong to the primitive tetragonal space group P41212, with unit cell parameters a = b = 100.64 Å and c = 216.24 Å. There are two

Novel Inhibition Mode of a Subtilisin molecules of the BASI–Savinase complex per asymmetric unit giving a Matthews coefficient39 of 2.77 Å3/Da, corresponding to a solvent content of ∼ 56%. The intensity data were indexed, integrated and scaled using the HKL2000 package with DENZO and SCALEPACK40 (Table 3). Structure solution and refinement The structure was solved by molecular replacement using the program PHASER,41 with the wild-type Savinase (PDB ID 1SVN) and BASI (PDB ID 1AVA) molecules as independent components in the rotation and translation parameter search. Both complexes of BASI–Savinase were clearly placed in the asymmetric unit, and the R-factors after the first 10 cycles of maximum-likelihood refinement by REFMAC542 were 25.8% and 33.6% for Rcryst and Rfree, respectively. The model was completed by visual inspection of the 2Fo − Fc and Fo − Fc electron density maps using the program Coot.43 Non-crystallographic symmetry averaging was used to improve the map, and non-crystallographic symmetry restraints were applied during the first steps of refinement. The R-factors converged to 23.3% and 28.1% for Rcryst and Rfree, respectively. At this stage, a number of water molecules were added to the model using Coot. The data were at first refined to 2 Å. In the final part of the refinement, the data set was reprocessed to 1.85 Å, but with the Rfree test set of reflections imported from the original 2-Å data set. This improved the electron density maps and helped rebuild the flexible loops. Following several steps of refinement, combined with visual checking of the model, Rcryst and Rfree dropped to 20.2% and 23.9%, respectively. The final model contains 6620 non-hydrogen protein atoms and 574 water molecules. Electron density peaks greater than the Table 3. Crystallographic data and refinement statistics Data collection Wavelength (Å) Space group Cell dimensions (Å)

1.0039 P41212 a = b = 100.64 c = 216.24 50–1.85 (1.92–1.85) 0.105 (0.471) 16.3 (1.6) 87.7 (41.4) 4.3 (1.6) 0.52 364,148 84,089

Resolution (Å) Rmergea I/σ(I) Completeness (%) Redundancy Mosaicity Total no. of reflections No. of unique reflections Refinement Rcryst Rfree Free R-value test set size (no reflections) (%) No. of non-hydrogen atoms No. of water molecules Mean B value (Å2) B value from Wilson plot (Å2) Bonds (Å) Angles (°)

0.202 0.239 5.0 6620 574 34.3 27.0 0.017 1.915

Ramachandran plot regions (%) Most favoured 88.5 Additionally allowed 11.4 Generously allowed 0.1 Disallowed 0.0 PP PP a Rmerge ¼ jIi ðh; k; lÞ  hIðh; k; lÞij= Ii ðh; k; lÞ. hkl i

hkl i

Novel Inhibition Mode of a Subtilisin 3σ level were identified as 4 Ca2+, 6 Na+ and 24 Cl− ions, all present in the crystallisation medium. The average B-factor for main chain atoms is 34.3 Å2. The refinement statistics are summarised in Table 3. The Ramachandran plot was defined by Procheck,44 and the distribution of residues is summarised in Table 3. Interface calculations The interface area of the complex between Savinase and BASI was obtained by evaluating the solvent-accessible area of A12 of the complex and then the solvent-accessible areas of A1 and A2, the surface areas of dissociated Savinase and BASI, respectively, and then computing B = (A1 + A2 − A12). The surfaces were computed analytically using the Lee and Richards algorithm28 and a probe radius of 1.4 Å. Inhibition of Savinase and proteinase K BASI wild type, F84V, R85G, A86T, Y87I, Y87A, T88V and T89A (seven concentrations ranging from 0 to 100 nM) were preincubated with Savinase or proteinase K (40 nM) for 10 min at room temperature in 50 mM glycine, pH 9.8, 150 mM KCl, 0.5 mM CaCl and 0.01% Triton X-100. N-SucAAPF-pNA (Sigma S 7388) was added (five concentrations ranging from 0.16 to 6.4 mM, in 250 μl). Changes in absorbance at 405 nM were measured and converted into initial rates of reaction. Km, Vmax and Ki were calculated for all BASI variants by non-linear regression; competitive inhibition was calculated using GraphPad prism, version 5.01 (GraphPad Software). PDB accession number The coordinates and structure factors have been deposited in the PDB under PDB ID 3BX1.

Acknowledgements This work was financially supported by the STF Program of the Danish Research Agency Ministry of Science, Technology and Innovation. We thank Lars Kobberøe Skov, Lars Beier and Søren Neve for helpful discussions and Allan Svendsen and Jürgen Carsten Franz Knötzel for proofreading of the manuscript. We also thank Lars Østergaard for valuable assistance with the enzyme kinetics.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2008.05.034

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