Eur. J. Biochem. 169,215-221 (1987) 0 FEBS 1987

The structure of a synthetic pepsin inhibitor complexed with endothiapepsin Jon COOPER, Steve FOUNDLING, Andrew HEMMINGS and Tom BLUNDELL Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, London

D. Michael JONES, Allan HALLETT and Michael SZELKE

Department of Chemical Pathology, Royal Postgraduate Medical School, London

(Received April 13/June 26, 1987) - EJB 87 0433

The conformation of a synthetic polypeptide inhibitor, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 0.20-nm resolution and refined to an agreement factor of 0.20. The inhibitor:

Pro Thr Glu Phe-R-Phe Arg Glu (R = -CH2NH-) is based on a chromogenic substrate of pepsin (EC 3.4.23.1). It has, in place of the scissile bond, a reduced peptide group which is resistant to hydrolysis and mimics the tetrahedral transition state. The inhibitor binds in an extended conformation with the reduced bond close to the essential aspartate side-chains of the enzyme. The hydrogen bonds and hydrophobic interactions between the enzyme and the inhibitor do not induce large conformational changes.

The aspartic proteinases, a family of enzymes including pepsin, are characterized by two essential aspartate residues (32 and 215 in pepsin) which have been shown by X-ray diffraction to reside at the active site [l -41. Pepsin is the major digestive enzyme of the stomach and like most aspartic proteinases is optimally active at low pH. Analysis of the kinetic parameters of hydrolysis, by pepsin, of a single peptide bond in small synthetic substrates has shown that the rate constants depend not only on the nature of the residues adjacent to the scissile bond, but also on several residues each side of the dipeptidyl unit indicating that aspartic proteinases have up to seven specificity subsites [5 , 61.

A study of the specificity of porcine pepsin [7] for tripeptides of the type : benzyloxycarbonyl-His-Xaa-Xaa- OMe showed that the highest k,,,lK, is obtained when both Xaa residues are phenylalanines. Varying the Xaas causes k,,, to change much more than K, indicating that tight binding at these subsites lowers the activation barrier of hydrolysis by stabilization of the transition state. A survey of the amino acids found to occur in the vicinity of pepsin-susceptible bonds in proteins [8] led to the design and synthesis of a useful chromogenic substrate nicknamed ‘Ralph’ based on a Phe- Phe dipeptide [9], Tables 1, 2.

The substrate has been converted to a potent pepsin in- hibitor by replacing the scissile bond with a reduced peptide group -CH2-NH- giving H-256 [4, 101 (Tables 1, 2) which inhibits endothiapepsin and porcine pepsin with Ki values of 60 nM and 40 nM respectively.

Correspondence to T. Blundell, Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, Malet Street, Lon- don, England WClE 7HX

Enzymes (IUB Recommendations, 1984). Endothiapepsin (EC 3.4.23.6); pepsin (EC 3.4.23.1); human renin (EC 3.4.23.15).

The structure of endothiapepsin has been solved at 0.21-nm resolution [ll]. The enzyme crystallizes in space group P21 with a unit cell: a = 5.36nm, b=7.4nm, c = 4.57 nm, B = 110”.

Endothiapepsin is a bilobal molecule and consists of 330 residues in 20 p strands and 4 small CI helices arranged in two topologically equivalent lobes which are related by an approximate twofold axis. Only 13 residues are identical in both lobes but 86 are in topologically equivalent positions. This indicates that the structure may have evolved by gene duplication, fusion and sequence divergence [12]. The catalytic site is in a large cleft (about 3.5 nm long) between the two domains. The two essential aspartates, which lie at the tips of conserved loops, are held close together by a network of hydrogen bonds [13]. In this arrangement the highly conserved residues next to both aspartates (Asp-Thr-Gly-Ser/Thr) form hydrogen bonds that restrain the carboxyls so that they are 0.29 nm apart and are approximately coplanar. This arrange- ment has a pseudo diad and the network of hydrogen bonds involving the threonines has been likened to a ‘fireman’s grip’ [13]. A solvent molecule is hydrogen-bonded symmetrically to both carboxyls (Fig. 1). Close to the aspartates the surface of the enzyme is predominantly hydrophobic. These residues make up the S3, S1 and S1‘ binding subsites, using the nomenclature of Schechter and Berger [14]. We now describe the growth of isomorphous cocrystals of endothiapepsin and H-256 at pH 4.6 and the solution of the inhibitor complex by the X-ray difference Fourier technique and least-squares refinement at 0.20-nm resolution to an agreement factor of 0.20.

MATERIALS AND METHODS The reduced Phe-Phe isostere of H-256 (Table 1) was syn-

thesized by reductive alkylation of the protected C-terminal

21 6

Table 1. A synthetic pepsin substrate and two inhibitors

Substrate/ P4 P3 P2 PI P1' P2' P3' inhibitor

'Ralph' Pro Thr Glu Phe -CO-NH- NOzPhe Arg Leu H-256 Pro Thr Glu Phe -CHZ--NH- Phe Arg Glu H-142 Fro His Fro Phe His Leu -CHz - NH- Val Ile His LYS

n I2l8 B

Fig. 1. A stereo view of the aspartate residues at the catalytic centre. The solvent molecule bound to both aspartates (W2) lies on the pseudo- twofold axis

Table 2. Kinetic data for 'Ralph' (from Hallett et al. [ 4 ] )

S - ' mM s - ' mM-'

Human pepsin 12 0.170 420

Endothiapepsin 12 0.003 4000 Porcine pepsin 89 0.080 1110

amino acid by the N-terminal aldehyde using sodium cyanoborohydride as reducing agent [lo]. The secondary amino group of the reduced bond was then protected with a 3,4-dichlorobenzyloxycarbonyl group and the C-terminal blocking group was removed for incorporation of the dipeptide analogue into the inhibitor molecule.

Cocrystals of endothiapepsin and H-256 were grown in acetate buffer at pH 4.6 by slowly dissolving a tenfold molar excess of inhibitor in a 2 mg/ml solution of enzyme followed by addition of ammonium sulphate to 55% saturation (2.2 M) and a few drops of acetone (method based on [15]). X-ray data were collected on seven crystals to 0.20-nm resolution using a Hilger and Watts Y290 and an Enraf-Nonius CAD4F diffractometer. A total of 30000 reflections were measured and corrected for diffraction geometry, fading and absorp- tion. These were merged to give 19600 unique reflections (representing 88% of the reflections in the 0.2-nm sphere) with an Rsym of 0.04. The reflections, IFp,[, were scaled to the native structure factors, IFPI, and electron density maps were calculated at 0.29 nm and, later, 0.24 nm with weighted coefficients: IFPIl - IFPI and 2 lFpll - lFpl [16] together with the refined native phase. These were displayed on an Evans and Sutherland PS2 using the program FRODO [17] as modified by Dr I. J. Tickle. A model of H-256 was then built into the difference electron density. These preliminary atomic coordinates were subjected to stereochemically restrained least-squares refinement using the program RESTRAIN [lS, 191. A few cycles of rigid body refinement were performed using the medium resolution data to compensate for a small

difference in the p angle of the inhibitor and native crystals. All atomic coordinates and isotropic temperature factors were then refined by alternating least-squares refinement with model building into weighted 2 F, - F, and F,, - F, maps [16]. A 2 F, - F, map for part of the inhibitor is shown in Fig. 2. In refinement the shifts applied to each atom were restrained to minimize deviation from ideal bond lengths, angle distances, non-bonded distances, chirality, peptide planes and side-chain planes. In every cycle each class of restraint was weighted accmding to the r.m.s. deviation from ideality of the whole molecule. The isotropic temperature factors of the inhibitor atoms were found, on average, not to be significantly different from those of the enzyme indicating that the inhibitor is present in unit occupancy. This was con- firmed by subsequent refinement of the occupancy which gave a figure of 94% for the inhibitor. The final R factor was 0.20 for reflections in the 2.0-nm to 0.2-nm range [F > 1 o (F)] and the r.m.s. coordinate error of the complex (calculated from [16]) was 0.024 nm. After refinement the program FITZ [20] was used to obtain a least-squares fit of the inhibitor complex onto coordinates of the native enzyme and other complexes to compensate for small rigid body movements during refine- ment and hence give a meaningful comparison of the con- formations.

RESULTS AND DISCUSSION

The electron density at 0.2 nm extends along the active- site cleft from subsites S4 to S3' and defines all the side- chains of H-256 from P4 to P3'. The reduced peptide bond, as anticipated, is close to the two essential aspartates. The phenylalanines at P1 and P1' lie in hydrophobic pockets on each side of the catalytic centre.

The backbone of H-256 is extended and is responsible for most of the hydrogen bonds to the enzyme. The main-chain torsion angles are shown in Fig. 3 together with the side-chain conformations using the notation of Janin et al. [21] who observe a preponderance of the gt conformation, as is the

21 7

Fig. 2. The 2 F, - F, electron density for the N-terminal side of the inhibitor (H-256) and the reduced bond

+I8C

PSI

C

.'; I : . I

' I

Fig. 3. A Ramachandran plot ,for the inhibitor. The conformation of each side chain (Xlangle) is indicated, g', trans to the carboxyl group; g-, trans to the hydrogen; t, trans to the amino group

case with H-256. The inhibitor makes two hydrogen bonds to the main chain of the active site 'flap' (Trp-71 to Gly-82) and four to the loops which contain the essential aspartates (32 and 215), Fig. 4. The main chain at P3 makes two hydrogen bonds with threonine-219. The hydroxyl of the Thr-219 acceDts a hvdrogen bond from the amino gram of P3 and the

carbonyl group of this residue accepts another hydrogen bond from the peptide nitrogen of 219. The carbonyls of the pseudo- diad-related and absolutely conserved glycines, 34 and 21 7, accept hydrogen bonds from the P2' and P1 amide nitrogens, respectively. The active-site flap makes two hydrogen bonds with the inhibitor involving an interaction between the carbonyl of Ser-74 and the amide nitrogen of P3', and another between the carbonyl of P1' and the amide nitrogen of glycine- 76. Hence the enzyme provides four hydrogen-bond acceptors and two donors. In the native structure five of these groups are hydrogen bonded to water molecules in the active site (Table 3). These, together with another 17 water molecules, are displaced when the inhibitor binds. The hydrogen-bonding interactions form a pattern that appears to be common to a number of inhibitor complexes [3, 4, 22-24]. The enzyme probably makes these hydrogen bonds to all substrates and inhibitors of sufficient length indicating that selectivity is de- termined by other interactions at the pockets. The increase in k,,,/K,,, with substrate length [5, 61 is probably due, in part, to the greater number of hydrogen bonds that can be formed. These bonds may be important in positioning the substrate correctly for catalysis and/or inducing strain in the scissile bond.

Two side-chains of the inhibitor form hydrogen bonds to the enzyme via water molecules. The threonine side-chain at P3 is hydrogen bonded to a water molecule (W108) which forms a hydrogen bond to a carboxyl oxygen of Asp-304. The electron density for W108 at 0.2 nm is elongated, indicating that it has an alternative location 0.2 nm from the refined position. A strong feature of positive F, - F, electron density provides additional evidence for this position in which the water molecule can form good hydrogen bonds with the hydroxyl at P3 of the inhibitor and a carboxyl oxygen of Glu-30. One guanidinium NH2 group of the arginine at P2' is 0.29 nm from a well-ordered water lW3441 which accents a

22 8 C I

Glym Ser 74

Fig. 4. A schematic diagram of the hydrogen bonds between H-256 and the active site of endothiapepsin

Table 3. Hydrogen bond donor-acceptor distances for (a) the main- chain of the inhibitor (H-256). ( b ) the subset of water molecules. displaced by the inhibitor, that are bound to the same groups of the enyzme

Group Hydrogen bonds to the inhibitor

Hydrogen bonds to waters in native endothiapepsin

nm OH-21 9 NH P3 0.27 NH-219 CO P3 0.27 CO-217 NH P1 0.28 NH-76 CO P1’ 0.30 CO-34 N H P2’ 0.29 (:O-74 NH P3’ 0.28

nm -

W302 0.32 W200 0.28 W242 0.33 W198 0.27 W343 0.28

hydrogen bond from the peptide nitrogen of Ser-74. The other NH2 group forms a weak hydrogen bond with the carbonyl of Leu-128.

In the initial difference map, small features of positive and negative density indicated a slight shift of the active site flap (Trp-71 -Gly-82) towards the inhibitor. This must be due to the hydrogen bonds and van der Waals interactions with H-256. The flexibility of the flap is due to the three glycines and six serines in the highly conserved sequence of 13 residues. The positions of the flap and the residues of the ‘fireman’s grip’ for the native enzyme and the refined inhibitor complex are shown in Fig. 5. The most noticeable conformational change is that in the flap although the movement is small and 1s certainly much less than the hinge-like rotation of 0.2 nm, at the tip of the flap, observed in the pencillopepsin-pepstatin complex [23]. The larger change in penicillopepsin may be due to disruption of several intermolecular hydrogen bonds that hold the flap more open in the native structure than in the inhibitor complex.

The hydrogen bonds that maintain the structure of the catalytic centre of endothiapepsin (the fireman’s grip) are not disrupted by the inhibitor because of the lack of con- formational change, although the aspartate carboxyls are less coplanar than in the native structure. This is probably due to a van der Waals contact between the C-p of the Phe at P1 and the outer carboxyl oxygen of Asp-32.

A comparison of the temperature factors of the active-site flap in the complexed and the native enzyme shows that the flap is less mobile or disordered when the inhibitor is bound

(Fig. 6). The large decreases in the temperature factors of Ser-74 and Gly-76 must be due to the hydrogen bonds to the inhibitor involving these residues. The lower thermal mobility of Tyr-75 may be due to an aromatic interaction with the phenylalanine at PI. Other residues that become less dis- ordered in the complex include those of the fireman’s grip and several binding pockets although the changes are much less convincing than for the active-site flap.

The reduced peptide bond lies close to both catalytic carboxyls. A negative peak in the original difference map indicates that the solvent molecule between the aspartates is displaced by the inhibitor. Aspartic proteinase activity ex- hibits a bell-shaped pH dependence in acid (e.g. [25]) and since the crystals were grown close to the optimal pH we consider that the aspartate diad will have an overall negative charge. In this environment the reduced peptide nitrogen is probably protonated and hence could form a salt link with the diad. Interestingly it has been found that a salt link between the amino group of a propart lysine residue and the two aspartates in pepsinogen may help to maintain the inactive state [26]. Another reason for the potency of H-256 may be that the reduced bond, lacking the restraints of a planar peptide group, allows an additional degree of flexibility in the P1-P1’ dipeptidyl unit similar to that of the transition state. This could allow the phenylalanines to interact maximally with the subsites. The rapid turnover by porcine pepsin of substrates containing two adjacent phenylalanine residues [7] may be due to the mode of binding that we observe in the inhibitor structure, Fig. 7. The preference of pepsins for hydrophobic residues at the P1 and PI’ positions is reflected in the large number of van der Waals interactions (< 0.4 nm) between non-polar residues and H-256 at these subsites. As shown in Table4 the residues that we identify at these pockets in endothiapepsin are largely conserved in porcine pepsin (se- quence from [27]).

We have compared the interactions of H-256 with those of H-142, a reduced peptide inhibitor designed to be specific for human renin (Table 1, 2) [28]. The bound structure of H- 142 has been solved at 0.21 nm with an R factor of 0.19. H-256 and H-142 inhibit endothiapepsin with Ki values of 60 nM and 160 nM respectively [4]. Table 5 shows the residues of the enzyme that make up the binding pockets for H-256 (contacts < 0.4 nm). Since both inhibitors form the same hydrogen bonds to the enzyme, the greater potency of H-256 relative to H-142 must be due to a larger number of van der Waals contacts. Clearly the Leu and Val side chains at PI and PI’ of H-142 cannot bind as tightly as the phenylalanines of

219

Fig. 5. A stereo view showing the active-site residues of the native and complexed enzyme as dashed and solid lines, respectively. The inhibitor lies horizontally in the cleft

= N a t l r e

T r p S e r I l e S e r T y r G l y A s p G l y S a r S e t S e r S e r G l y

Fig. 6. The change in temperature factors of the active-site flap. The average isotropic temperature factors are plotted against residue number for the native enzyme (solid bars) and inhibitor complex (blank bars)

Table 4. Residues at the primary specificity sites of endothiapepsin and their equivalents in porcine pepsin

Site Endothia- Porcine pepsin pepsin

s1 ASP-30 Tyr-I5 Phe-111 Leu-I 20

S1’ Ile-213 Ile-297 Ile-299 Ile-301

Ile TYr Phe Tle

Ile

Leu Ile

GlY

H-256. At the S1’ pocket the Phe ring of H-256 makes contact with an interesting cluster of isoleucines namely 297,299 and 301 in a strand that crosses the loop containing aspartate- 21 5. However, these isoleucines are too far from the P1’ valine of H-142 to form van der Waals interactions. The Phe at P1 of H-256 makes van der Waals contacts with Tyr-75 of the active-site flap, Phe-1 11 and Leu-120. The pocket is large and is not completely filled even by the phenylalanine (Fig. 7). It has been found that replacing the Phe at P1 of a substrate with a bulkier and more flexible residue, e.g. cyclohexylalanine gives a further enhancement in binding to porcine pepsin [7]. It has been suggested that the side chain hydroxyl of Tyr-75, a constituent of this pocket, might act as a proton donor for the nitrogen of the scissile bond because of the proximity of this residue to the aspartate diad [29]. However, this mecha-

nism has been withdrawn [30] and the H-256 complex clearly shows that the phenolic hydroxyl group points away from the catalytic centre and that the tyrosine ring interacts with the side chain at PI. The hydroxyl of Tyr-75 also forms hydrogen bonds with the indole nitrogen of Trp-39 and with Ser-35 via a solvent molecule.

There appears to be a significant difference in the positions of the reduced bond atoms of H-142 and H-256. The nitrogen in H-142 is closer to the aspartate diad. This may be because the van der Waals interactions in the S1 and S1’ pockets are weaker than in the H-256 complex causing the electrostatic interaction with the negative charge of the aspartates to dominate. These differences are shown in Fig. 8. Interestingly it has been found that renin substrates containing phenyl- alanines at P1 and P1‘ are non-hydrolyzable and yet act as competitive inhibitors [31]. This indicates that the phenyl- alanines dominate the interactions so much that the scissile bond is not positioned correctly for hydrolysis. The Ki values of these inhibitors can be improved by several orders of magnitude by replacing the scissile bond with a reduced isostere [28] and by the addition of a hydrophobic residue to fill the S 5 pocket [32]. However, H-256 is a relatively poor inhibitor of human renin (Ki > 7600 nM). The absence of a residue at P5 of H-256 and the presence, in renin, of hydro- phobic side chains at all subsites, except S2 [33], probably accounts for the low potency of this inhibitor, which has a high proportion of polar side chains.

The residues at P4, P3, P2’ and P3’ of H-256 occupy similar positions to those of H-142. The Glu at P2 of H-256 takes up a slightly different orientation to that of the histidine in H-142, which is closer to Asp-77. The greater length of the arginine side chain at P2’ of H-256 enables it to explore parts of the binding pocket unavailable to the smaller isoleucine residue in H-142. The aliphatic moiety of the arginine is sandwiched between the active-site flap (Ile-73 and Ser-74) and Leu-128, and the guanidinium group is exposed to solvent where it forms a hydrogen bond with the enzyme via a water molecule. The active-site flap makes many additional van der Waals contacts with H-256. For example, the side chain of Asp-77 makes several contacts with the phenylalanine at P1 and the glutamate at P2, as does Tyr-75 with the aromatic ring of PI. The carboxy-terminal glutamate of the inhibitor (P3’) curls out from beneath the flap and is half exposed to solvent in a similar position to the penultimate histidine residue of

The phenylalanine rings of H-256 interact with a number of polar side chains. The Phe at P1 is within van der Waals contact distance of aspartates 30, 77 and 32 and the P1’ side chain forms intramolecular contacts with the carboxyls of the glutamates at P2 and P3‘, and the essential aspartate 215. It

H-142.

220

Fig. 7. A stereo view of H-256 and the active site cleft

Fig. 8. A stereo view of the bound structures of H-256 (-) and H-I42 (----) shown superposed with the active-site carboxyls

Table 5. The subsites of endothiapepsin as defined by interactions with H-256

s 4 ASP-12

53 Asp-12 Thr-219

Thr-218 s 2 Tyr-75

s1 ASP-30 ASP-71 Leu-I 20

Ile-213 Ile-297

Leu-128

S1' ASP-32

S2' Gly-34

S3' Ser-I4

Thr-219

Ala-I 3

Gly-76 Tyr-222

Ser-79 ASP-32

ASP-21 5

Gly-34 ASP-21 5 Ile-299

Ile-73 Thr- 130

Gly-76

ASP-77

ASP-77

Tyr-75 Phe-111 Gly-217

Gly-76 Thr-218 Ile-301

Ser-74 Phe- 189

can be seen (Fig. 7) that in all these interactions the phenyl- alanines adopt orientations such that the neighbouring carboxyl groups, which are presumably protonated, are close to the edges of the rings. An edgewise contact between an aromatic ring and an oxygen atom is the most favourable owing to the partial positive charge on the hydrogen atoms at the edge of the ring resulting from the C-H dipole moment [34]. It has been suggested that interactions between buried oxygens and aromatic rings make a significant contribution to the stability of protein structures.

This study has given some insight into the physical mecha- nism of proteolysis by aspartic proteinases. The active-site flap of endothiapepsin is rather disordered and/or mobile in the native structure as revealed by the relatively high isotropic temperature factors. The flap must be flexible to allow the substrate to gain access to the active site. When bound, a substrate, like the inhibitor would make contact both with the flap and with the loops containing the aspartates via hydrogen bonds and van der Waals interactions. Accordingly the tem- perature factors for the flap atoms are lower in the inhibitor complex than in the native structure. However, some mobility of the flap is necessary to allow the products to leave. The displacement of a number of strongly bound solvent molecules in the active site must compensate, to some extent, for the loss of entropy when the substrate binds. It has been suggested that a decrease in mobility of part of an enzyme, upon substrate binding, would decrease the stability of the enzyme-substrate complex. This is advantageous to catalysis because it reduces the drop in free energy that occurs on formation of the complex between enzyme and unreacted substrate and hence lowers the activation barrier [35,36]. Specificity can, therefore, be achieved without excessively tight binding of substrate. The reduction in disorder of the active-site flap indicates that such a mechanism may operate in aspartic proteinases. The absence of significant conformational change in the H-256 complex indicates that an induced fit model is un- likely.

In catalysis the formation of the transition state probably involves nucleophilic attack at the carbonyl carbon by a water molecule activated by the aspartate diad [13, 301. The mechanism is not thought to involve an acyl-enzyme inter- mediate because of the failure to trap covalently bound sub- strate [37]. Only when the tetrahedral intermediate has formed

221

could the phenylalanines adopt the conformation that we observe in the inhibitor structure. This indicates that in the enzyme-substrate complex the Phe side chains could not bind so tightly to the S1 and S1’ pockets as in the transition state and that the binding energy of the P1 and P1‘ side chains is used to distort the substrate or stabilize the tetrahedral intermediate. Distortion of the scissile peptide bond from planarity would increase the susceptibility of the carbonyl carbon to nucleophilic attack by decreasing the delocalization of electrons. However, in renin the effect of phenylalanines at P1 and P1’ is to stabilize the enzyme-substrate complex, causing competitive inhibition. This must be due to differences in the shape and composition of the primary specificity pockets.

This work has been performed with the financial support of the Science and Engineering Research Council and the Medical Research Council (UK). We would also like to acknowledge Dr J. Kay (Univer- sity College, Cardiff) and Dr B. Dunn (University of Florida, Gainsville) for making available the kinetic data, Dr S. P. Wood and Dr L. H. Pearl for useful discussions, and Dr I. J. Tickle for the implementation of computer programs at Birkbeck College.

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