6
Protein Science (1996). 5:2453-2458. Cambridge University Press. Printed in the USA. Copyright 0 1996 The Protein Society Strategy to design peptide inhibitors: Structure of a complex of proteinase K with a designed octapeptide inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2 at 2.5 A resolution AJAY K. SAXENA,’ TEJ P. SINGH,’ KLAUS PETERS? SIEGFRIED FITTKAU? AND CHRISTIAN BETZEL3 ‘Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India ’Institute of Physiological Chemistry, Martin Luther University Halle-Wittenberg, Medical Faculty, 06097 Halle (Salle), Germany ’Institute of Physiological Chemistry, C/O DESY Notkestrasse 85, 22603 Hamburg, Germany (RECEIVED June 25, 1996; ACCEFTED September 5, 1996) Abstract The crystal structure of a complex formed by the interaction between proteinase K and a designed octapeptide amide, N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2, has been determined at 2.5 8, resolution and refined to an R-factor of 16.7% for 7,430 reflections in the resolution range of 8.0-2.50 8,. The inhibitor forms a stable complex through a series of hydrogen bonds and hydrophobic interactions with the protein atoms and water molecules. The inhibitor is hydro- lyzed between Phe41 and DAla” (I indicates the inhibitor). The two fragments are separated by a distance of 3.2 8, between the carbonyl carbon of Phe4’ and the main-chain nitrogen of DAla”. The N-terminal tetrapeptide occupies subsites S1-S5 (S5 for acetyl group), whereas the C-terminal part fits into Sl’-S5’ region (S5’ for amide group). It is the first time that such an extended electron density for a designed synthetic peptide inhibitor has been observed in the prime region of an enzyme of the subtilisin family. In fact, the inhibitor fills the recognition site completely. There is only a slight rearrangement of the protein residues to accommodate the inhibitor. Superposition of the present octa- peptide inhibitor on the hexapeptide inhibitor studied previously shows an overall homology of the two inhibitors, although the individual atoms are displaced significantly. It suggests the existence of a recognition site with flexible dimensions. Kinetic studies indicate an inhibition rate of 100% by this specifically designed peptide inhibitor. Keywords: conformation; inhibition rate; octapeptide inhibitor; proteinase K; X-ray structure Proteinase K isolated from fungus Tritirachium album Limber (Ebel- ing et al., 1974) is a serine protease of the subtilisin family. It consists of 279 amino acids with a molecular weight of 28,790 Da (Jany et al., 1986). It can readily hydrolyze native proteins and remains active in the presence of urea and SDS (Rauber et al., 1978; Jany & Nitsche, 1983). These properties make proteinase K a useful tool for the preparation of protein-free samples of DNA or RNA (Wiegers & Hilz, 1971, 1972; Gross-Bellard et al., 1973). It displays no significant specificity toward protein substrates, but its cleavage pattern on oxidized insulin B-chain suggests that the smallest peptide to be hydrolyzed by proteinase K should be a tetrapeptide (Kraus et al., 1976). Furthermore, the enzymes of the subtilisin family are used as additives in washing detergents (Van Reprint requests to: T.P. Singh, Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110029, India; e-mail: tps@ medinst.ernet.in. der Lann et al., 1995). Therefore, it is of high interest to define the binding region completely for useful protein engineering and also to demonstrate the design principle of specific peptides (Bromme et al., 1986; Singh et al., 1990, 1995; Singh & Kaur, 1996). So far, five complexes of proteinase K and designed peptides have been analyzed. Three of them with synthetic (1) Z-Ala-Ala-chloromethyl ketone (Betzel et al., 1986) (where Z denotes carbobenzoxyl), (2) Z-Ala-Phe-chloromethyl ketone (Betzel et al., 1988a), and (3) methoxysuccinyl-Ala-Ala-Pro-Ala-chloromethyl ketone (Wolf et al., 1991) showed that the ketone group was covalently bound to the enzyme through the serine and histidine residues of the active site, and the peptide portion forms the third strand of antiparallel P-pleated sheet with the twostrands of the recognition site in proteinase K. The overall geometry of the enzyme remains un- changed on inhibitor binding in these complexes. The inhibition rate of (2) was found to be higher than that of (1) (28:10), sug- gesting the preference for Phe at the specificity pocket SI (termi- 2453

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Protein Science (1996). 5:2453-2458. Cambridge University Press. Printed in the USA. Copyright 0 1996 The Protein Society

Strategy to design peptide inhibitors: Structure of a complex of proteinase K with a designed octapeptide inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2 at 2.5 A resolution

AJAY K. SAXENA,’ TEJ P. SINGH,’ KLAUS PETERS? SIEGFRIED FITTKAU? AND CHRISTIAN BETZEL3 ‘Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India ’Institute of Physiological Chemistry, Martin Luther University Halle-Wittenberg, Medical Faculty, 06097 Halle (Salle), Germany

’Institute of Physiological Chemistry, C/O DESY Notkestrasse 85, 22603 Hamburg, Germany

(RECEIVED June 25, 1996; ACCEFTED September 5, 1996)

Abstract

The crystal structure of a complex formed by the interaction between proteinase K and a designed octapeptide amide, N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2, has been determined at 2.5 8, resolution and refined to an R-factor of 16.7% for 7,430 reflections in the resolution range of 8.0-2.50 8,. The inhibitor forms a stable complex through a series of hydrogen bonds and hydrophobic interactions with the protein atoms and water molecules. The inhibitor is hydro- lyzed between Phe41 and DAla” (I indicates the inhibitor). The two fragments are separated by a distance of 3.2 8, between the carbonyl carbon of Phe4’ and the main-chain nitrogen of DAla”. The N-terminal tetrapeptide occupies subsites S1-S5 (S5 for acetyl group), whereas the C-terminal part fits into Sl’-S5’ region (S5’ for amide group). It is the first time that such an extended electron density for a designed synthetic peptide inhibitor has been observed in the prime region of an enzyme of the subtilisin family. In fact, the inhibitor fills the recognition site completely. There is only a slight rearrangement of the protein residues to accommodate the inhibitor. Superposition of the present octa- peptide inhibitor on the hexapeptide inhibitor studied previously shows an overall homology of the two inhibitors, although the individual atoms are displaced significantly. It suggests the existence of a recognition site with flexible dimensions. Kinetic studies indicate an inhibition rate of 100% by this specifically designed peptide inhibitor.

Keywords: conformation; inhibition rate; octapeptide inhibitor; proteinase K; X-ray structure

Proteinase K isolated from fungus Tritirachium album Limber (Ebel- ing et al., 1974) is a serine protease of the subtilisin family. It consists of 279 amino acids with a molecular weight of 28,790 Da (Jany et al., 1986). It can readily hydrolyze native proteins and remains active in the presence of urea and SDS (Rauber et al., 1978; Jany & Nitsche, 1983). These properties make proteinase K a useful tool for the preparation of protein-free samples of DNA or RNA (Wiegers & Hilz, 1971, 1972; Gross-Bellard et al., 1973). It displays no significant specificity toward protein substrates, but its cleavage pattern on oxidized insulin B-chain suggests that the smallest peptide to be hydrolyzed by proteinase K should be a tetrapeptide (Kraus et al., 1976). Furthermore, the enzymes of the subtilisin family are used as additives in washing detergents (Van

Reprint requests to: T.P. Singh, Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110029, India; e-mail: tps@ medinst.ernet.in.

der Lann et al., 1995). Therefore, it is of high interest to define the binding region completely for useful protein engineering and also to demonstrate the design principle of specific peptides (Bromme et al., 1986; Singh et al., 1990, 1995; Singh & Kaur, 1996). So far, five complexes of proteinase K and designed peptides have been analyzed. Three of them with synthetic (1) Z-Ala-Ala-chloromethyl ketone (Betzel et al., 1986) (where Z denotes carbobenzoxyl), (2) Z-Ala-Phe-chloromethyl ketone (Betzel et al., 1988a), and (3) methoxysuccinyl-Ala-Ala-Pro-Ala-chloromethyl ketone (Wolf et al., 1991) showed that the ketone group was covalently bound to the enzyme through the serine and histidine residues of the active site, and the peptide portion forms the third strand of antiparallel P-pleated sheet with the two strands of the recognition site in proteinase K. The overall geometry of the enzyme remains un- changed on inhibitor binding in these complexes. The inhibition rate of (2) was found to be higher than that of (1) (28:10), sug- gesting the preference for Phe at the specificity pocket SI (termi-

2453

Page 2: inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2

2454 A.K. Saxena et al.

nology after Schlechter & Berger, 1967). Furthermore, the inhibition rate of (3) was found to be much higher than (2) (88:28), indicating that the saturation of the principal recognition site (subsites Sl-S5) enhances the binding affinity of the inhibitor. The fourth was a molecular complex with N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-NH2, which showed the highest inhibition rate so far at 95% (Betzel et al., 1993). The peptide is hydrolyzed. The tetrapeptide fragment is held in the main recognition site with Phe at the specificity subsite S 1, whereas the dipeptide fragment is located in the prime region of the enzyme. This was the first complex that indicated an important role of the prime region in the binding to substratel inhibitors. Therefore, we extended the length of the peptide from hexapeptide to octapeptide N-Acetyl-Pro-Ala-Pro-Phe-DAla-Ala- Ala-Ala-NH2 to explore the full dimensions of the prime region in the enzyme. We report here the structure of a complex of protein- ase K and an octapeptide N-Acetyl-Pro-Ala-Pro-Phe-DAla-Ala- Ala-Ala-NH2. Recently, a ternary complex of proteinase K with Hg2+ and a substrate analogue hexapeptide amide (Saxena et al., 1996) and a complex with its naturally occurring protein inhibitor, PKI3 from wheat-germ (Pal et al., 1994), have been reported.

Results and discussion

It is well known from earlier crystallographic studies on proteinase K (Pahler et al., 1984; Betzel et al., 1988b) that the substrate recognition site is formed by two peptide chains A ~ n ~ ~ - T y r ' " and Ser'32-Gly'36. The recognition site residues are accessible to the solvent on one side, whereas the other ends of these strands ter- minate at the catalytic triad of A ~ p ~ ~ - H i s ~ ~ - S e r ~ ' ~ . The catalytic residues display low thermal motion with a well-defined geometry and are hardly accessible to solvent molecules. Beyond the hydro- lytic triad of A ~ p " - H i s ~ ~ - S e r ~ * ~ lies the prime region, which, unlike the principal recognition site, is not characteristically de- fined but appears to be filled with several solvent water molecules.

The structure of the complex has been refined using stereochem- ically restrained least-squares procedure to an R-factor of 16.7% for all the data in the resolution range of 8.0-2.5 A. Luzzati plots (Luzzati, 1952) of the R-factor as a function of resolution are consistent with average maximum coordinate errors approximately 0.19. A SIGMA analysis (Read, 1986) gives a similar value of about 0.17 A. The structure contained 2,022 protein atoms, 53 inhibitor atoms, and 206 water molecules. The mean temperature factor for all the main-chain atoms of the protein was 1 1.6 A2 and for all the side-chain atoms 12.1 A'. For the peptide, the mean temperature factor for all main-chain atoms was 26.3 A2 and for all side-chain atoms 23.9 A2. The RMS error in bond lengths was 0.018 A, compared to the target value of 0.01 A. The torsion angles (0) of the peptide planes have a mean deviation of 3.0" with respect to the ideal value of 180". Additional statistics are given in Table 1.

As seen from the difference electron density map (2F, - 2Fc) in Figure 1, the peptide molecule is fitted into the electron density, which extends to the other end of the prime region. It is the first clear evidence of an extended electron density in the S'-region. The hydrolyzed octapeptide fills the recognition site completely, extending its two ends to the surface of the enzyme molecule (Fig. 2), thus making it a perfect complex with a designed syn- thetic peptide inhibitor. The distance calculations show that the peptide inhibitor is held by several intermolecular hydrogen bonds (Table 2). It is a stable complex and the inhibitor is hydrolyzed as in the previous complex (Betzel et al., 1993) where the electron

Table 1. Data collection, data processing, and refinement de- tails for the proteinase K-inhibitor complex

Space group P432 I 2 Cell dimensions a = b = 68.0, c = 107.7 8, X-ray source Sealed tube X-ray generator,

operating at 50 kV, 50 mA (focalsize 1.2 X 0.4 mm2)

A = 1.5418 A Radiation CuKdgraphite monochromator;

Collimation 0.4 X 0.4 mm2 Detector MAR Research imaging

Crystal to plate distance 100 mm Maximum resolution 2.5 8, Total no. of observed reflections 49,646 No. of independent reflections 7,647 Completeness of data up to 2.5 8, 83% Overall merging Rsym(I) factor 6.9% Deviation from ideal bond length 0.01 8 Deviation from ideal bond angles 2.6" Deviation of planar groups 0.031 8, Deviation of chiral volume 0.21 A ? Deviation of the peptide plane 3.0" Final R-factor for 7,430 reflections 16.7%

Mean temperature factor B 13.4 A2

plate scanner

in the resolution range 8.0-2.5 8,

density showed discontinuity at the scissile bond. Both the cleaved fragments move apart to a distance of 3.2 8, and remain anchored to their respective binding sites. A detailed examination of inter- molecular forces between the inhibitor and the enzyme molecule indicates that the residue Phe4' at PI subsite and the P1' subsite residue DAla" seem to play significant roles in the binding of the inhibitor to the enzyme. It is noteworthy that a large number of water molecules are involved in stabilizing the complex.

Interactions between the enzyme and the inhibitor

As seen from the schematic drawing in Figure 3, the N-terminal fragment forms an antiparallel &strand between the two parallel protein strands of A ~ n ~ ~ - T y r ' ~ and Ser'32-Gly"6, whereas the C-terminal fraction slides into the water-rich environment. The carbonyl oxygen atom of the acetyl group forms a hydrogen bond with a water molecule Watm2. The Pro"(0) is involved in two hydrogen bonds with Wat600 and Wat6", where Watm' is linked to the carbonyl oxygen of Glyio2 through a strong hydrogen bond. The Ala2'(N) and Ala2'(0) form hydrogen bonds with G l ~ ' ~ ~ ( 0 ) and GIY"~(N), respectively, as observed in the arrangement of antiparallel @-strands. Pro"(0) forms a hydrogen bond with A ~ n ' ~ ' ( N 6 2 ) .

The most extensive interactions are observed involving Phe4' of the inhibitor and Ser224(0y) of the enzyme. The carbonyl carbon of Phe4' is in a tetrahedral configuration because the distance to Ser224(0y) is 2.4 A, which is less than the expected van der Waals distance but much longer than the covalent C to 0 bond length of a true intermediate. The corresponding distance in the complex with a hexapeptide is 2.1 A (Betzel et al., 1993). Thus, the Ser224(0y) retains some characteristics of a substrate analogue tetrahedral intermediate. This has also been observed in the bovine

Page 3: inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2

Structure of proteinase K and designed octapeptide 2455

11 I I 21 "

L I .

Fig. 1. Stereo view of the difference electron density (2F0 - 2FC)acalc for the peptide N-Ac-Pro-Ala-Pro-Phe-DAla-(Ala)-,-NH2 contoured at 2.1 u.

trypsin inhibitor-trypsin complex (Huber et al., 1974), and also in a study of a subtilisin-eglin C complex (Bode et al., 1987). How- ever, such an evidence of cleavage in the complex of proteinase K and its natural inhibitor PKI3 from wheat has not been reported (Pal et al., 1994). In addition, the Ser224(0y) is at hydrogen bond forming distances from Phe41(N), DAla5'(N), and H i ~ ~ ~ ( N e 2 ) (Table 2; Fig. 3). The carbonyl oxygen of Phe41 forms two hydro- gen bonds with Thr223(Oyl) and Ser224(N). The extensive inter- actions observed above indicate that the present octapeptide is a suitably designed inhibitor of proteinase K.

The C-terminal tetrapeptide DAla-Ala-Ala-Ala-NH2 occupies the Sl'-S5' subsites and is stabilized predominantly by hydrogen bonds involving a series of water molecules and main-chain atoms; the side-chain H-bonding is not possible because the sequence has all Ala residues (Table 2; Fig. 3). The C-terminal fragment remains anchored through two terminal hydrogen bonds involving DAla5'(N) and C-terminal NH with Ser224(0y) and Asn6'(0), respectively. These interactions determine the separation of 3.2 A between the hydrolyzed peptide fragments. The corresponding distance in the previous complex was found to be 3.07 A. From the nature of these interactions, it appears that the peptide in the prime region is placed in a void filled with solvent water molecules. Because the region is highly hydrophilic, the C-terminal sequences with small side chains such as Ala seem to be good peptide inhibitors of proteinase K (Betzel et al., 1988a). In addition, there exist several

hydrophobic interactions between the peptide and enzyme, which are listed in Table 2.

Movement of protein on inhibitor binding

The superposition of the refined coordinates of proteinase K chain from the inhibited and native crystals was done by least-squares fitting using a program LSQKAB from CCP4 package (1979). It gives an R M S discrepancy of 0.6 8, for the C a atoms and 0.8 8, for all atoms. This is to be compared with estimated errors in the coordinates of the native (0.14 A) and inhibitor complex (0.19 A) given above. In very few parts of the polypeptide chain are there significant differences in the C a position between the native en- zyme and the inhibitor complex. The RMS deviation for the re- gions containing the active-site and the binding site residues between the native enzyme and the inhibitor complex after refinement is approximately 0.6 A.

Thus, there is no evidence of a major conformational reorien- tation of the proteinase K polypeptide chain upon binding of the inhibitor. The overall impression is that the active site is poised to accept an inhibitor or substrate and undergoes relatively small changes when such molecules are bound. The largest shifts in C a positions on inhibitor binding are of 0.8 A for active site His69 and -0.7 8, for GlylW, Ser"', Leu133, Gly134, and G l ~ l ~ ~ , which make part of the recognition site. When compared with

Fig. 2. View of the inhibitor peptide placed on the top of recognition site. The hydrolyzed peptide is shown in yellow. The peptide fills the binding site completely, extending its two arms to the surface of the protein.

Page 4: inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2

A . K . Saxena et a!.

Table 2. Hydmgen bond lengths and van der Waals distances between the inhibitor and proteinase m a t e r molecules

Inhibitor atom Proteinase K/water

Ac(O) Watml Pro l I (0) Watm' Pro l I (0) Wat MKI

Ala 2I(N) G l ~ ' ~ ~ ( 0 ) Ala 2I(O) G I Y ' ~ ~ ( N ) Pro 31(0) AsnI6'(N82) Phe 4I(N) Ser2z4(0y) Phe 4I(O) nr223(oy1) Phe 4I(O) SerZZ4(N) D-Ala 5I(N) ~ e r * ~ ~ ( ~ y ) D-Ala 5I(O) WatsI2 Ala 6I(O) Wat479 Ala 7I(O) Ala 7I(O) Wats30 Ala 81(0) Wat542 Ala 8I(O) Wat4"J NH2 Asn6'(0)

Distances (A)

3.0 3.0 2.8 3.1 3.0 3.1 3. I 2.8 2.7 3. I 3.0 3. I 3.0 3.1 3.0 2.9 3.0

Between water molecules involved in inhibitor binding Warm1 Gly'02(0) 3.0

Enzyme atoms involved in the catalytic mechanism ~ e r * ~ ~ ( ~ y ) H i ~ ~ ~ ( N e 2 ) 2.8 A ~ p ~ ~ ( 0 8 2 ) H i ~ ~ ~ ( N 6 1 ) 2.8

Distance of active Ser*"(Og) to the carbonyl carbon atom

~ e r ' ~ ~ ( O y ) Phe4'(C) 2.4

Distance of Phe 4I(C) to the amide nitrogen atom of D-Ala 51 Phe 41 (C) D-Ala 51 (N) 3.2

Van der Waals distances (<3.2 A) between the inhibitor

of the scissile bond

and proteinase K Distance

Inhibitor atom Proteinase K 4, Ala 21 (Cp) Ala 21 ( C a ) Phe 41(Ce2) Phe 4I(Ce2) Phe 4I(Cn) Phe 4I(O) Phe 4I(O) Phe 4I(C) Phe 4I(O) D-Ala 5I(N) D-Ala 5I(O) Ala 6I(Cp) Ala 8I(Cp) Ala 8I(Cp) Ala SI(Cf3)

2.9 3.0 2.9 3.0 2.9 3 .O 2.9 2.9 3.0 2.9 3.0 2.9 3.2 3 .O 3.0

other synthetic inhibitor complexes of proteinase K (Betzel et al., 1986, 1988a, 1993; Wolf et al., 1991; Saxena et al., 1996), the displacements observed in the present complex are more pro- nounced. The side chains of several charged residues on the sur- face of the protein were found to be displaced substantially when compared with the native protein (Betzel et al., 1988b). This may be due partly to the presence of 30% ethanol as solvent.

As seen from Figure 4, the octapeptide Ac-Pro-Ala-Pro-Phe- DAla-Ala-Ala-Ala-NH2 in the present complex has been super- imposed on the hexapeptide Ac-Pro-Ala-Pro-Phe-DAla-Ala-NH2 from the previous complex (Betzel et al., 1993). Both the peptides are cleaved and their overall placements in the enzyme are very similar, but the individual atoms are displaced substantially. The largest displacement corresponds to 3.3 A for DAla"'.

Conclusions The structure of a complex of proteinase K with a designed sub- strate analogue octapeptide inhibitor has been determined. This is the first time that an extended electron density of a designed pep- tide inhibitor has been observed in the prime region of the enzyme (Fig. I). In fact, this inhibitor fills the binding region completely (Fig. 2 ) . The Phe residue of the peptide is held by hydrophobic forces in the SI-subsite, whereas the two arms of the peptide saturate the SI-S5 and SI'-S5' subsites, respectively (Fig. 2). The important implications are: (1) a fully characterized binding region of the enzyme exists, ( 2 ) the Phe residue must occupy the S I - subsite for an effective, and ( 3 ) the principal and the prime rec- ognition sites must be saturated completely by the peptide. The octapeptide inhibitor is hydrolyzed, but does not fully dissociate, and the distance between scissile carbonyl carbon of Phe4' and Serzz4(0y) is 2.4 A. The hydrolyzed fragments move apart to a distance of 3.2 A. The N-terminal fragment aligns in the middle of two noninteracting parallel @-strands of the protein by forming an antiparallel @-strand. The C-terminal tetrapeptide is held like an extended string by two anchor-like terminal hydrogen bonds (Fig. 3). In previous complex with a hexapeptide, the prime region was incompletely filled. However, a least-squares superimposition of the hexapeptide on the present octapeptide indicates a similar placement of the two inhibitors although individual atoms show substantial displacements (Fig. 4). The results of the present com- plex define the recognition site completely, including the prime region, and also establish the design strategy using peptide models.

It clearly suggests that the lengths of two peptide segments on either side of the scissile bond should be at least of the order of four residues to saturate the principal and prime regions of the binding site. Both should be in the extended conformations. Sub- stitution of Phe at the PI subsite and DAla at the PI ' subsite are very critical. The Phe residue fills the large hydrophobic pocket, whereas DAIa provides a desirable conformation to place the pep- tide appropriately in the binding region.

Materials and methods Proteinase K was obtained from Merck (Darmstadt, Germany) and purified by Gel filtration on a Sephadex G-75 column in 50 mM Tris-HCI, pH 7.5, containing I mM CaCI2. Fractions of highest activity were pooled, dialyzed exhaustively at 4 "C against 1 mM calcium acetate, and lyophilized.

The acylated octapeptide amide was synthesized by coupling of Ac-Pro-Ala-Pro-Phe-OH and H-DAla-(Ala)3-NH2 using the mixed anhydride method. Ac-Pro-Ala-Pro-Phe-OH was prepared from Ac-Pro-Ala-Pro-OH synthesized according to Thompson and Blout (1973) and H-Phe-OBzl.HC1 as described by Bromme et al. (1986). H-DAla-(A1al3-NH2 was obtained by deprotection of N-carbobenzoxy-derivative, which was prepared by coupling of Z-DAla-(Ala)3-OH and H-Ala-NH2. Z-DAla-(Ala)2-OH was syn- thesized using standard procedures and H-Ala-NH2 from the free amino acid methyl ester according to Chambers and Carpenter

Page 5: inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2

Structure of proteinase K and designed octapeptide 2451

/"; Asn mi

Fig. 3. Schematic drawing of the complex and atom numbering as described in the text. Hydrogen bonds are indicated by dashed lines. S5-S4-S3-S2-S1-Slf-S2'-S3'-S4'-S5' define the subsites according to Schlechter and Berger (1967); the corresponding numbering scheme on the inhibitor is P1 for Phe4', P2 for Pro3', P3 for Ala", P4 for Pro", and P5 for the acetyl group, and on the prime region P1' for DAlaS1, P2' for Ala6', P3' for Ala", P4' for Ala", and P5' for N H 2 .

( 1955). All steps of the synthesis procedure were checked by HPLC and TLC, respectively, as well as by determination of melting points and optical activities of the intermediate products. The struc- ture and the molecular weight of the final octapeptide derivative were confirmed by H-2D-NMR and amino acid analysis after a total hydrolysis and mass spectrometry.

The effect of the peptide as inhibitor on proteinase K was in- vestigated by kinetic assay with N-succinyl-(Ala)3-CO-NH-(C&)- NO2 as a substrate. The substrate was purchased from Sigma Chemical Co. (USA). For inhibition studies, the inhibitor was dissolved 0.5 p d p L in 50 mM Tris.HC1, 5 mM CaCI2, pH 8.0, containing 30% ethanol. Stock solutions of proteinase K (0.2 pgI pL) and of the substrate s~ccinyl-(Ala)~-CO-NH-(C~H~)-N0~ (1 mM) were prepared in the same buffer without ethanol; in all experiments, the molar ratio of inhibitor to enzyme was 25: 1. Assays contained 0.7 nmol of proteinase K, 17.5 nmol of inhibitor, 0.75 pmol of substrate, 0.05-5% ethanol. After enzyme and in-

hibitor were incubated for 15 min at 25 "C, the substrate succinyl- (Ala),-C0-NH-(C&)-NO2 was added and allowed to react for 1 h. The reaction was then stopped with glacial acetic acid and the released p-nitrophenylolate monitored at 410 nm with a Kontron UVIKON-810 spectrophotometer. The optical density at 410 nm compared well with the optical density for the substrate at similar concentration, suggesting a 100% inhibition. More detailed studies were reported earlier on a series of peptides including the present octapeptide (Bromme et al., 1986).

The purified proteinase K was cocrystallized with octapeptidea- mide A~-Pro-Ala-Pro-Phe-DAla-(Ala)~-NH~. The lyophilized en- zyme was dissolved at 10% (w/v) in 50 mM Tris-HC1, 1 mM CaCI2, pH 6.5, with twofold molar excess of the inhibitor; 25-pL drops of this solution were equilibrated in the sitting drop vapor diffusion method against 1 M NaN03 in the same buffer at 16 "C. Single crystals of size 0.5 X 0.4 X 0.4 m m 3 grew within 3-4 days. For X-ray intensity data collection, one crystal was mounted in a

Fig. 4. Least-squares fitting of the octapeptide from the present complex and the hexapeptide from the earlier complex (Betzel et al., 1993).

Page 6: inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2

245 8 A.K. Saxena et al.

glass capillary. The X-ray intensities were collected to 2.50 A resolution at 4 "C using a MAR Research Imaging Plate Scanner mounted on a sealed tube X-ray generator with a graphite mono- chromated CuKa radiation. The crystal buffer contained 30% eth- anol. The intensity data were restricted to 2.50 A resolution because the crystals of proteinase K containing ethanol tend to diffract slightly poorly (unpubl. results). Crystallographic data, data col- lection, and processing details are given in Table 1. Data were processed using the MOSFLM program system (Leslie et al., 1986).

The crystals of the complex are isomorphous with those of native proteinase K. For refinement and interpretation of the elec- tron density, the coordinates of the native proteinase K refined to 1 .50 A were used (Betzel et al., 1988b) in the traditional difference Fourier methods to determine the binding modes of the peptide inhibitor. Water molecules located in the active site region of the structure were removed prior to the refinement. The refinement was performed by restrained parameter least-squares analysis using PROLSQ/ PROTIN program (Hendrickson & Konnert, 1981) with fast Fourier routines to compute structure factors and gradients (Agarwal, 1978). Initially, we used the model structure along with two Ca2+ atoms and water molecules for 15 cycles of x y z refine- ment against the low-resolution shell (8.0-3.0) .& When the re- finement converged at 3.0 A, higher angle data were included in a few shell-wise expansions (8.0-2.8) A, (8.0-2.6) A, and (8.0- 2.5) A, with five additional cycles of xyz and individual B-factor refinements. During the refinement, adjustment of side chains and water molecules was performed by inspecting ( 2 F , - F,.) and (2F, - 2Fc) difference Fourier syntheses. The density of the in- hibitor was clearly visible in the region of active site. The peptide was built into the electron density. There was a clear discontinuity in the electron density beyond Phe4I, which suggested that the octapeptide was hydrolyzed. To establish the conformation and geometry of the hydrolyzed bond between Phe4' and DAla", sev- eral omit maps, where the surrounding region was omitted from the phase calculations, were inspected. All the graphics work was performed using 0 program package (Jones et al., 1985) on an INDIGO silicon graphics system. Finally, the model was refined using stereochemically restrained least-squares procedure to an R-factor of 16.7% for all the data in resolution range 8.0-2.5 A (7,430 reflections with no u cut off). Luzzati plots (Luzzati, 1952) of the R-factor as a function of resolution are consistent with average maximum coordinate errors of approximately 0.19. A SIGMA analysis (Read, 1986) gives similar value of about 0.17 A. Refinement statistics are summarized in Table 1. The refined co- ordinates will be deposited in the Brookhaven Protein Data Bank.

Acknowledgments

This work was supported in part by a grant from the Council of Scientific and Industrial Research, New Delhi (India).

References

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