Crystal structure of yellow meal worm α-amylase at 1.64 Å resolution1

J. Mol. Biol. (1998) 278, 617±628

Crystal Structure of Yellow Meal Worm a -Amylase at Ê Resolution 1.64 A Stefan Strobl1, Klaus Maskos2, Michael Betz2, Georg Wiegand2 Robert Huber2, F. Xavier Gomis-RuÈth2* and Rudi Glockshuber1* 1

Institut fuÈr Molekularbiologie und Biophysik, EidenoÈssische Technische Hochschule HoÈnggerberg, CH-8093, ZuÈrich Switzerland 2 Max-Planck-Institut fuÈr Biochemie, D-82152 Planegg-Martinsried, Germany

The three-dimensional structure of the a-amylase from Tenebrio molitor larvae (TMA) has been determined by molecular replacement techniques Ê; using diffraction data of a crystal of space group P212121 (a ˆ 51.24 A Ê Ê b ˆ 93.46 A; c ˆ 96.95 A). The structure has been re®ned to a crystallographic R-factor of 17.7% for 58,219 independent re¯ections in the 7.0 to Ê resolution range, with root-mean-square deviations of 0.008 A Ê for 1.64 A bond lengths and 1.482 for bond angles. The ®nal model comprises all 471 residues of TMA, 261 water molecules, one calcium cation and one chloride anion. The electron density con®rms that the N-terminal glutamine residue has undergone a post-transitional modi®cation resulting in a stable 5-oxo-proline residue. The X-ray structure of TMA provides the ®rst three-dimensional model of an insect a-amylase. The monomeric enzyme exhibits an elongated shape approximately Ê  46 A Ê  40 A Ê and consists of three distinct domains, in line with 75 A models for a-amylases from microbial, plant and mammalian origin. However, the structure of TMA re¯ects in the substrate and inhibitor binding region a remarkable difference from mammalian a-amylases: the lack of a highly ¯exible, glycine-rich loop, which has been proposed to be involved in a ``trap-release'' mechanism of substrate hydrolysis by mammalian a-amylases. The structural differences between a-amylases of various origins might explain the speci®city of inhibitors directed exclusively against insect a-amylases. # 1998 Academic Press Limited

*Corresponding author

Keywords: a-amylase; X-ray structure; yellow meal worm; Tenebrio molitor; a/b-barrel

Introduction a-Amylases (a-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a family of endo-amylases that catalyze the hydrolysis of a-D-(1,4)-glucan linkages in starch components, glycogen and various other related carbohydrates. They play a central role in carbohydrate metabolism of animals, plants, and microorganisms. Moreover, a-amylases have become one of the most valuable enzymes in biotechnology, especially in the food and starch processing industry (Vihinen & MaÈntsaÈlaÈ, 1989). Present address: F. X. Gomis-RuÈth, Centre d'Investigacions i Desenvolupament (C.S.I.C.), Carrer Jordi Girona, 18-26, 08034 Barcelona, Spain. Abbreviations used: ANA, Aspergillus niger a-amylase; BAA, barley a-amylase; BLA, Bacillus licheniformis a-amylase; CGT, cyclodextrin glycosyl transferase; PPA, porcine pancreatic a-amylase; r.m.s., root-mean-square; TMA, Tenebrio molitor a-amylase. 0022±2836/98/180617±12 $25.00/0/mb1667

Many insects, especially those that feed on grain products during larval and/or adult life, depend on their amylases for survival. This is particularly true for the beetle Tenebrio molitor, a cosmopolitan pest of stored products. The catalytic properties of the grub's a-amylase (TMA) have been investigated before (Buonocore et al., 1976a; Strobl et al., 1997), as well as its inhibition by several inhibitors in vitro (Applebaum, 1964; Shainkin & Birk, 1970; Silano et al., 1973; Buonocore et al., 1976b; Silano & Zahnley, 1978; Buonocore et al., 1980; GarcõÂaMaroto et al., 1991) and in vivo (Applebaum, 1964). In contrast to mammals, the yellow meal worm, i.e. the larva of T. molitor, expresses only a single a-amylase, TMA, consisting of 471 amino acid residues with a molecular mass of 51.3 kDa. We have recently determined the complete amino acid sequence of this monomeric protein by cloning its cDNA (Strobl et al., 1997). TMA is an acidic protein with a calculated pI of 4.3. The pH optimum for # 1998 Academic Press Limited

618

Structure of Yellow Meal Worm -Amylase

the cleavage of starch was determined to be 5.8 (Buonocore et al., 1976a), which means that the a-amylase is well adapted to its physiological environment in the larval midgut, where a slightly acidic pH of 6.4 is prevalent (Srivastava & Srivastava, 1961). The enzymatic mechanism of a-amylases is very complex and not yet completely elucidated. Neither is inhibition by the widely occurring natural a-amylase inhibitors. Up to now, all structural studies on the interaction of a-amylases with substrate analogs (Qian et al., 1995; Casset et al., 1995), carbohydrate inhibitors (Qian et al., 1994; Machius et al., 1996) and proteinaceous inhibitors (Wiegand et al., 1995; Bompard-Gilles et al., 1996) have been performed with the pig pancreatic enzyme (PPA). Although the amino acid sequences of mammalian and insect a-amylases are very similar (Strobl et al., 1997), several inhibitors of insect a-amylases have been reported to inhibit PPA and other mammalian a-amylases only to a very limited extent or not at all (Shainkin & Birk, 1970; Silano et al., 1973, 1975; Buonocore et al., 1977; GutieÂrrez et al., 1990; Chagolla-LoÂpez et al., 1994; for a review, see Richardson, 1990). Recently, we described the puri®cation of TMA to homogeneity and crystallization in the form of well-ordered orthorhombic crystals of space group P212121 (Strobl et al., 1997). Here, we report the three-dimensional structure determination using molecular replacement techniques and subsequent Ê resolution. This is the ®rst re®nement to 1.64 A reported structure of an insect a-amylase. With knowledge of the structures of mammalian salivary (Ramasubbu et al., 1996) and pancreatic a-amylases (Qian et al., 1993; Larson et al., 1994; Brayer et al., 1995; Machius et al., 1996), the three-

dimensional structure of TMA will provide insight into the structural basis for the species speci®city of a-amylase inhibitors. Moreover, the structure of an a-amylase from a grain-consuming insect might facilitate the design of inhibitors exclusively directed against insect a-amylases, thus being of interest in phytopharmacy.

Results and Discussion Quality of the structure and identification of post-translational protein modifications The ®nal model of TMA rendered a crystallographic R-factor of 17.7% (free R-factor 20.6%) for 58,219 unique re¯ections in the resolution range Ê . The statistics of the data between 7.0 and 1.64 A collections and re®nement are given in Table 1. The ®nal 2Fo ÿ Fc electron density map shows continuous density at the 1 s level for all mainchain atoms except for those of Asp446, for which continuous density is observed at the 0.8 s level. A Ramachandran plot (Ramachandran & Sasisekharan, 1968) of the f/c angles indicates that 87.2% of the amino acid residues are in the most favoured regions and 12.4% are in the additionally allowed regions. Asp390 is localized in a generously allowed region and Ser12 is the only residue in a disallowed region, as de®ned by the program PROCHECK (Laskowski et al., 1993). However, the side-chains of both latter residues are well de®ned by the electron density map like those of most other residues. Exceptions are the surface-exposed side-chains of Glu135, Glu378 and Gln381 (disordered beyond Cg), of Asp389 (disordered beyond Cb), Asp446 (disordered beyond Ca), and Lys470 (disordered beyond Cd). A single cispeptide bond was observed between residues

Table 1. Data collection and re®nement statistics of free TMA Diffraction data Space group Ê) Cell constants (A No. of measurements No. of unique reflections (I > 3s(I)) Average multiplicity Rmergea Ê) Completeness (%) (20±1.64 A Ê) (1.69± 1.64 A Refinement and final model Ê) Resolution range (A Reflections used Crystallographic R-factor b Free R-factor r.m.s. deviation from target values for Ê) Bonds (A Angles ( ) Ê 2) Bonded B-factors (A No. of (non-hydrogen) protein atoms Ê 2) Aver. B-factor for protein atoms (A Ions Calcium (KA 501) Chloride (CI 502) Solvent molecules a b

Rmerge ˆ (hj|I(h)j ÿ < I(h) > I)/(hj|I(h)jI) R-factorˆ (hkI||Fobs| ÿ |Fcalc||)/(hkI|Fobs|)

P212121 a ˆ 51.24; b ˆ 93.46; c ˆ 96.95 244,244 58,219 4.2 0.057 99.9 99.2 7.0±1.64 55,053 0.177 0.206 0.008 1.482 1.479 3606 15.3 Ê 2) 1 (B-factor 11.79 A Ê 2) 1 (B-factor 2.01 A 261

Structure of Yellow Meal Worm -Amylase

619

Figure 1. Stereo view of the electron density of the N-terminal pyroglutamate and the following lysine residue of TMA The electron density was calculated with coef®cients (2Fobs ÿ Fcalc) and contoured at 1.0 s.

Val123 and Pro124. The electron density clearly con®rms the post-translational modi®cation of the N-terminal glutamine residue to 5-oxo-proline (Figure 1), which had been indicated by biochemical experiments (Strobl et al., 1997). A single potential N-glycosylation site was identi®ed in a solventexposed loop of TMA at Asn447 (Asn447± Gly448± Ser449), but mass spectrometry of puri®ed TMA as well as the electron density map proved that the enzyme does not bear a sugar moiety. Eight of the nine cysteine residues of TMA are involved in non-overlapping disul®de bonds (Cys28 ± Cys84, Cys134± Cys148, Cys354 ±Cys360, and Cys425± Cys437). The free thiol of Cys241 is only partially solvent accessible. Overall structure The ®nal three-dimensional model of TMA consists of a single polypeptide chain of 471 amino acid residues, one calcium ion, one chloride ion and 261 water molecules. TMA displays an elongated overall shape with dimensions of Ê  46 A Ê  40 A Ê . The enzyme approximately 75 A consists of three distinct domains, A (residues 1 to 97 and 160 to 379), B (residues 98 to 159) and C (residues 380 to 471). The domain architecture is in full agreement with the known models of mammalian (Qian et al., 1993, 1994, 1995; Larson et al., 1994; Brayer et al., 1995; Wiegand et al., 1995; Machius et al., 1996; Ramasubbu et al., 1996), plant (Kadziola et al., 1994), fungal (Boel et al., 1990; Brady et al., 1991; Swift et al., 1991) and bacterial a-amylases (Machius et al., 1995; Hwang et al., 1997). All four cystine bridges of TMA are intradomain disul®des. A representation of the overall polypeptide fold, as well as the location of the bound ions and the residues presumably involved in catalysis, is given in Figure 2. Table 2 presents the positions of secondary structure elements. Domain A Domain A, the dominating three-dimensional unit of TMA, is composed of two segments, residues 1 to 97 and 160 to 379. It forms an eightstranded, parallel b-barrel, which is embraced by a concentric circle of eight helical segments (seven

a-helices and one 310-helix), thus displaying the fold of a (b/a)8-barrel. Additional secondary structure elements are located in the loops of the barrel. Although, the (f/c) angles of two residues, Ser12 in b-strand 1 (see above) and Gly38 in b-strand 2 are not in the b-sheet region, both residues disturb the geometry of the b-sheet structure to only a limited extent. The hydroxyl groups of Ser12 and Thr320 (b-strand 8) form an additional direct hydrogen bond and the backbone amide groups of both residues form a further watermediated hydrogen bond. Both interactions are compatible with the geometry of the b-sheet structure. The symmetry of the central (b/a)8-barrel is interrupted by domain B, which is inserted between the third b-strand and the third a-helix of domain A. Protruding loops of varying length are observed after b-strand 2 (containing an additional

Figure 2. Ribbon plot of TMA. Domain A is shown in blue, domain B in green, and domain C in red. The chloride anion and the calcium cation are represented by a purple and a yellow sphere, respectively, and the active-site residues Asp185, Glu222, and Asp287 are depicted in pink.

620

Structure of Yellow Meal Worm -Amylase

Table 2. Sequence positions of the three domains of TMA and their regular secondary structure elements A. Domain A (b/a)8-barrel (residues 1 to 97 and 160 to 379)a Ab1 Asn11±Leu16 Aa1 Trp21±Phe31 Trp56±Tyr60 Ab2 Phe37±Ile42 A3102a Aa2b Glu74±Ala87 Ab3 Arg90±Ala95 Aa3 Asp161±Leu177 Ab4 Gly181±Asp185 Aa4 Pro192±Gly201 Ab5 Phe218±Glu222 A3105 Lys233±Tyr236 Ab6 Cys241±Leu243 Aa6a Phe245±Gln256 A3106b Leu261-Asn266 Ab7 Ala279± Val281 Aa7a Asp287±Gly292 Aa7b Pro302±Ala314 Ab8a Thr319±Ser324 Ab8b Gly347±Asn349 Ab8c Thr353±Ser355 Aa8 Arg365±Ala376 B. Domain B (residues 98 to 159) Bb1 Gly105±Gly107 Bb2 Ser111-Asp114 Bb3 Asn119±Tyr120 Bb4 Tyr125±Gly126

Ba1

Ala142-Asn147

C. Domain C: Greek-key motif/b-sandwich (residues 380 to 471)b Cb1 Val382±Ser387 Cb2 Gln392±Arg397 Cb3 Gly401±Thr406 Cb4 Leu411±Asn416 Cb5 Gly422±Asp426 Cb6 Glu431±Ser433 Cb7 Ser436±Cys437 Cb8 Lys440± Val444 Cb9 Ser449±Leu454 Cb10 Val462±His466 a Secondary structure elements not involved in the formation of the (b/a)8-barrel are in italics. b Secondary structure elements not involved in the formation of the b-sandwich structure are in italics.

310-helix), a-helix 4, b-strand 5, a-helix 6 (containing another 310-helix), b-strand 7 (containing an additional a-helix), and b-strand 8 (containing two short antiparallel b-strands), respectively. It is a general feature of (b/a)8-barrel enzymes that the loops on the C-terminal side of the b-barrel, connecting b-strands to a-helices, are more complex than those of the N-terminal side. They contain the active site residues and those involved in ligand binding (Machius et al., 1995). Domain B Domain B, inserted into domain A between the third b-sheet and the third a-helix, starts at residue 98 and ends at residue 159. It is formed by several extended segments and a short a-helix. Four short extended segments consisting of two to four residues line up to form an irregular antiparallel b-sheet. The a-helix extends from Ala142 to Asn147 and contains one of the residues, Arg146, involved in calcium binding. A helix at a topologically equivalent position was also described for a-amylases from mammalian and fungal origin and in cyclodextrin glycosyl transferase, but not in bacterial (Machius et al., 1995) or plant a-amylases (Kadziola et al., 1994). Domain B forms a cavity against the b-barrel of domain A in which the calcium ion is bound. The

cation is of fundamental importance for the structural integrity of the molecule (Vallee et al., 1959, Buonocore et al., 1976a; Vihinen & MaÈntsaÈlaÈ, 1989; Violet & Meunier, 1989; Machius et al., 1995). On a structural level, domain B is the least conserved segment among a-amylases from different origins and other carbohydrate-processing enzymes (Machius et al., 1995). Nevertheless, segment B always folds into a globular domain, which provides ligands for the calcium ion at the interface with domain A. Domain C Domain C is located exactly opposite to domain B on the other side of domain A (Figure 2). No contact exists between domains B and C. The C domain comprises the C-terminal residues 380 to 471 and forms a separated folding unit, exclusively made up by b-sheets. Eight of the ten strands fold into a b-sandwich structure with ``Greek key'' topology (Richardson, 1981). In addition, domain C contains an insertion between b-strands 5 and 8 that consists of a separate two-stranded anti-parallel b-sheet. The b-sandwich fold of the C domain is also displayed by C domains of a-amylases from other origins (Machius et al., 1995) and by the corresponding domains of cyclodextrin glycosyl transferases (Klein & Schulz, 1991; Kubota et al., 1991), but not the C domain of barley a-amylase (BAA), which is much shorter and consists only of a ®ve-stranded anti-parallel b-sheet (Kadziola et al., 1994). However, b-strands 1, 2, 3 and 10 in TMA are topologically equivalent with b-strands 1, 2, 3 and 5 in BAA, and TMA's b-strand 5 approximately superimposes with BBA's b-strand 4 (Figure 3). To summarize, the ``lower'' part of the b-sandwich in the C domain of TMA is replaced by a single b-strand and some loops in BAA, and the inserted two-stranded b-sheet present in TMA (b-strands 6 and 7) is completely absent from the C domain of BAA. The dimensions of the C domains of both a-amylases are about the same, except for the distance perpendicular to the antiparallel b-sheet at the interface to domain A Ê  19 A Ê for TMA and (approximately 31 A  27 A Ê Ê Ê 19 A  24 A  15 A for BAA). As clearly depicted in Figure 3, the course of the backbone of the polypeptide chain at the interface of the two domains A and C remains conserved. The conservation of the interface of the two domains among a-amylases (and cyclodextrin glycosyl transferases) suggests an important role for enzyme activity, stability, folding and/or substrate binding. Active site and substrate-binding cleft The enzymatic mechanism of a-amylases is very complex and not yet completely elucidated. Three acidic side-chains, belonging to two aspartic acid and one glutamic acid residue, are directly involved in catalysis. This assignment is based on structural studies of enzyme/inhibitor complexes

621

Structure of Yellow Meal Worm -Amylase

Figure 3. Superposition of the domain A/domain C interface of TMA (green) and barley a-amylase (BAA) (red). Only the trace of the TMA residues 251 to 326 and 359 to 471 and of the BAA residues 251 to 403 are shown. TMA residues 263 to 266, 304 to 315, 366 to 377, 390 to 406 and 460 to 467 were superimposed on BAA residues 262 to 265, 303 to 314, 333 to 344, 359 to 375 and 395 to 402.

(Qian et al., 1994; Wiegand et al., 1995; Machius et al., 1996) and mutagenesis experiments (Sùgaard et al., 1993; Takase, 1994). On the basis of sequence homology and structural comparison we propose that Asp185, Glu222 and Asp287 of TMA, which are located at the bottom of the domain A b-barrel,

take part in catalysis. Corresponding residues could be detected in related enzymes sharing the proposed catalytic mechanism (Table 3: for a review, see Svensson, 1994). The polysaccharidebinding groove of porcine pancreatic a-amylase (PPA) accommodates at least six sugar units

Table 3. Conservation of a-amylase side-chains involved in catalysis and binding of substrate and ions TMA Subsite 3 3 5 4 3,4 3 3,4

PPA*

BLA

ANAa

BAA

CGT

Residues at and around the active site H99 D185 K188 H189 E222 H286 D287

H101 D197 K200 H201 E233 H299 D300

R183 N285 R321

R195 N298 R337

N98 R146 D155 H189

N100 R158 D167 H201

H105 D231 K234 H235 E261 H327 D328 Residues involved R229 N326 E300

H122 D206 K209 E210 E230 H296 D297

H92 D179 K182 G183 E204 H288 D289

H140 D229 K232 H233 E257 H327 D328

in chloride bindingb R204 R177 N295 N287 I326 ±

R227 N326 A356

Residues involved in calcium bindingc N104 N121 N91 d E162 D138 D200 D175 D148 H235 E210 G183 A141

N139 I190 D199 H233

Non-conserved residues are written in bold letters, residues involved in catalysis are underlined. TMA, Tenebrio molitor a-amylase; PPA, porcine pancreatic a-amylase; BLA, Bacillus licheniformis a-amylase; ANA, Aspergillus niger a-amylase; BAA, barley a-amylase; CGT, cyclodextrin glycosyl transferase. a PPA was chosen as a representative of mammalian a-amylases and ANA as a representative of fungal a-amylases. b For BLA, ANA, BAA and CGT the authors did not report electron density corresponding to the chloride ion. The residues listed are topologically equivalent to the residues involved in chloride binding in TMA. In the BAA structure, the third coordinating residue is replaced by a water molecule. c Calcium is not present in the BLA structure. The residues listed are topologically equivalent to the residues involved in calcium binding in TMA. In BAA, there are three calcium-binding sites. The one homologous to those of the other a-amylases is differently coordinated. d The corresponding residue in BLA is not visible in the electron density.

622 (Machius et al., 1996), the cleavage taking place between the third and the fourth pyranose. On the basis of crystal structures and model building, a reaction mechanism for the cleavage of glycosidic bonds by PPA was suggested (Mazur et al., 1994). This mechanism accounts for the importance of all three acidic side-chains and postulates a nucleophilic attack of a water molecule on C1 of the sugar moiety bound at subsite 3. Accordingly, this results in a linearization of the pyranose, followed by the cleavage of the polysaccharide chain. After cleavage, the aldehyde and hydroxyl groups of sugar unit 3 react intramolecularly to form again a cyclic hemiacetal. This model comprises no covalent intermediate between the enzyme and the substrate. The substrate-binding groove that accommodates six hexose units is covered by residues belonging to both domains A and B. All residues of PPA, which have been shown to interact with carbohydrate inhibitors (Qian et al., 1994; Machius et al., 1996), are conserved in the TMA model, except those of the PPA ¯exible ``glycine-rich'' loop (His305 and Gly306 in PPA), which is deleted in TMA. A comparison of TMA with a-amylases of mammalian, plant, bacterial and fungal origin and with cyclodextrin glycosyl transferases shows that within this enzyme family only ®ve residues of those interacting with the substrate analogs in PPA are completely conserved (Table 3): the three catalytic residues (Asp185, Glu222 and Asp287 in TMA) and two histidine residues (His99 and His286 in TMA). A lysine (Lys188) and another histidine (His189) residue are conserved in all but plant and Aspergillus niger a-amylases. The two completely conserved histidine residues and the two catalytic aspartic acid side-chains coordinate the sugar moiety at subsite 3, while that at subsite 4 is ®xed in its position by the catalytic glutamic acid, the second catalytic aspartic acid residue (Asp287) and the almost completely conserved His189. The lysine residue interacts with the C2 and C3-hydroxyl groups of the sugar molecule bound to subsite 5. Since we assume that there is a common mechanism within the a-amylase family for substrate recognition and discrimination between non-reducing and reducing ends, we suggest that this is directly accomplished by the residues at subsite 3. Evidence has been obtained that domain C provides a second binding site for starch, which is independent of the substrate-binding groove at the catalytic site. Two additional saccharide-binding sites distinct from the active site became evident from the X-ray structure analysis of PPA crystals soaked with substrate. One of the binding sites was found at the domain A/domain C interface accommodating a maltose molecule, the other on the surface of domain B nearby the calcium ion, binding a glucopyranose molecule (Qian et al., 1995). In a PPA/carbohydrate inhibitor complex acquired by co-crystallization of PPA with V-1532, the maltose-binding site was also detected, but not

Structure of Yellow Meal Worm -Amylase

Figure 4. Space-®lling models of porcine pancreatic aamylase (left molecule) and TMA (right molecule). Residues in porcine pancreatic a-amylase involved in substrate binding around the catalytic site (W58, W59, Y62, Q63, H101, Y151, V163, D197, A198, K200, H201, E233, I235, L237, E240, H299, D300 H305 and G306) and around the maltose-binding site at the domain A/ domain C interface (F315, K322, T376, C378, W382, V383, R387, W388, R389, E390 and R392) are depicted in red and green, respectively. Homologous residues in the TMA model are shown in the same colours (W56, W57, Y60, Q61, H99, Y139, V151, D185, A186, K188, H189, E222, I224, L226, E229, H286 and D287 in red; Y299, K306, M352, C354, Y358, V359, R363, W364, R365 and Q366 in green).

the glycopyranose binding site (Machius et al., Ê 1996). The former is located approximately 35 A away from the catalytic site (Figure 4). The presence of a second substrate-binding site is also suggested by inhibition experiments performed with a carbohydrate and a proteinaceous inhibitor (Alkazaz et al., 1996; Maskos et al., 1996), since the obtained inhibition pro®les will be explained best if PPA possesses at least one additional oligosaccharide-binding site. Mutation studies on Bacillus stearothermophilus a-amylase also suggest that substrate is bound at the interface of domains A and C (Holm et al., 1990), although the possibility cannot be ruled out that domain C might be involved in folding and/or stabilization of the whole enzyme. Chemical modi®cation and mutation of BAA isozymes 1 and 2 have led to similar results. Two tryptophan residues at the interface of the A and the C domains appear to be involved in binding starch granules, but one of them also seems to be critical for folding and/or stability (Gibson & Svensson, 1987; Sùgaard et al., 1993). The calcium-binding site All X-ray structures of a-amylases solved up to now contain a calcium ion at a conserved position (Table 3), the only exception being calciumdepleted Bacillus licheniformis a-amylase (BLA).

623

Structure of Yellow Meal Worm -Amylase

Figure 5. Coordination of the calcium ion in the TMA structure. The calcium ion is represented by a yellow sphere and water molecules by blue spheres. B domain residues are depicted in red and the A domain residue in green. Coordinating interactions are shown as blue lines.

However, the removal of the calcium ion in BLA causes local disorder around the Ca2‡-binding site, resulting in an inactive enzyme (Machius et al., 1995; Hwang et al., 1997). The Ca2‡-binding site in TMA is located at the interface of the domain A central b-barrel and domain B, on the same side of the b-barrel as the catalytic center. Its architecture, as depicted in Figure 5, is even conserved in distantly related a-amylases (Brayer et al., 1995; Machius et al., 1995; Table 3) and can be regarded as a distorted pentagonal bipyramid as originally described for the A. niger enzyme (Boel et al., 1990). The calcium ion is eight-fold coordinated to four amino acid residues (Asp155 is participating in calcium binding in a bidentate mode) and three water molecules. Distances are given in Table 4. In TMA, three residues of domain B participate in calcium binding via their side-chain oxygen atoms (Asn98 Od1 and Asp155 Od1 and Od2) and their backbone carbonyl oxygen atom (Arg146). The only residue of domain A in contact with the calcium ion, His189, is also coordinated via its carbonyl oxygen atom. His189 is conserved in all a-amylases, except for those of plant origin, where it is replaced by glycine (Table 3). The reason for the almost complete conservation of this histidine residue may be its role in interacting with the fourth sugar unit of the saccharide substrate accommodated in the active site. His189 forms a direct hinge between the catalytic site and the calcium-binding site. Chloride-binding site In contrast to the Ca2‡-binding site of a-amylases, a chloride-binding site has so far been detected only in a-amylases of mammalian origin (Qian et al., 1993, 1994, 1995; Larson et al., 1994; Brayer et al., 1995; Wiegand et al., 1995; Machius et al., 1996; Ramasubbu et al., 1996). However, a region equivalent to the chloride-binding site exists in the fungal (Brady et al., 1991; Swift et al., 1991, plant (Kadziola et al., 1994) and bacterial enzymes (Hwang et al., 1997; Table 3). In these structures, the position of the anion is replaced by a solvent molecule. Chloride allosterically activates PPA (Thoma et al., 1971; Levitzki & Steer, 1974; Yamamoto et al.,

1988), and is has been shown that the same is true for TMA (Buonocore et al., 1976a). The binding site for the anion is located on the same side of the b-barrel as the catalytic and the calcium-binding site, in the vicinity of both. In TMA, the chloride ion is coordinated to three amino acid residues of the A domain and one water molecule in a hemispherical grouping (Figure 6; Table 4). It is bound to NZ1 and NZ2 of Arg321 and to NZ2 and Ne of Arg183 in bidentate modes, and to Nd2 of Asn285 in a monodentate fashion. The second hemisphere of the chloride ion is shielded by hydrophobic groups, involving the methyl Cd1 of Leu243, the phenyl Cz of Phe245, and the methylene Cg of Glu222. Since Glu222 is one of the catalytically active residues, the chloride ion is directly linked to the active site. The chloride ion is located only Ê away from the water molecule that has been 4.5 A suggested to initiate the cleavage of the substrate chain (Mazur et al., 1994). The nucleophilicity of this water molecule might be enhanced by the negative charge of the anion.

Table 4. Coordination of the calcium and chloride ion in TMA Acceptor

Ê) Distance (A

Asn98 OD1 Arg146 O Asp155 OD1 Asp155 OD2 His189 O SOL511 SOL521 SOL530

Ca2‡ Ca2‡ Ca2‡ Ca2‡ Ca2‡ Ca2‡ Ca2‡ Ca2‡

2.4 2.4 2.5 2.6 2.4 2.4 2.6 2.4

Arg183 NE Arg183 NH2 Asn285 ND2 Arg321 NH1 Arg321 NH2 SOL522

Clÿ1 Clÿ1 Clÿ1 Clÿ1 Clÿ1 Clÿ1

3.3 3.3 3.1 3.3 3.2 3.0

Glu222 CG Leu243 CD1 Leu243 CD2 Phe245 CZ

Clÿ1 Clÿ1 Clÿ1 Clÿ1

4.4 3.6 4.5 3.1

Donor

624

Structure of Yellow Meal Worm -Amylase

Figure 6. Coordination of the chloride ion in the TMA structure. The chloride ion is represented by a purple sphere and the water molecule by a blue sphere. Residues coordinating the chloride anion are depicted in red, hydrophobic groups shielding the second hemisphere of the ion are shown in green, and the catalytic residue Glu222 in dark green. Coordinating interactions are shown as blue lines.

Structural comparison with mammalian a -amylases Insect a-amylases are closely related to mammalian a-amylases, as re¯ected by the high level of sequence identity (53.8%) between TMA and PPA (Strobl et al., 1997). The close relationship to the mammalian enzymes is also demonstrated by the fact that the three-dimensional structure of TMA could be solved by molecular replacement with the coordinates of unliganded PPA (Machius et al., 1996), but not with the coordinates of a-amylases from plant, fungal or bacterial origin (see Materials and Methods). In TMA, domain A is shorter by 14 residues, domain B by ten residues and domain C by one residue than the corresponding domains in PPA. The positions of the secondary structure elements are basically the same as those in PPA (Larson et al., 1994) and the distances between the calcium and the chloride ions are almost identical Ê in TMA and 17.1 A Ê in in both structures (16.9 A a PPA). The C atoms of 410 structurally conserved residues superimpose with a root-mean-square Ê (Figure 7(a)). deviation of 2.2 A The TMA structure is especially distinguished from the structures of the mammalian a-amylases by the lack of three loops in the vicinity of the active site (Figure 7). In the absence of substrate, a ¯exible loop in domain A of the mammalian enzymes (residues 304 to 310 in PPA) adopts an ``open'' position, as observed in the structures of PPA (Machius et al., 1996), human salivary a-amylase (Ramasubbu et al., 1996) and human pancreatic a-amylase (Brayer et al., 1995), and undergoes a reorientation upon binding of carbohydrate inhibitors with r.m.s. movements of the Ê , and side-chain His305 main-chain atoms of 3.5 A Ê atoms of 4.5 A, respectively (Qian et al., 1994; Machius et al., 1996). His305 and Gly306 of PPA interact with the carbohydrate inhibitor subunits 2 and 6 bound to the main substrate-binding groove. It was suggested that this glycine-rich

loop may act as a gateway for substrate binding and could be involved in a kind of ``trap-release'' mechanism in substrate hydrolysis (Ramasubbu et al., 1996). This should be re¯ected in a signi®cant improvement of the catalytic parameters compared to a closely related a-amylase lacking the ¯exible loop. In TMA, this loop is actually shorter by the three amino acid residues (Figure 7(b)) that undergo the largest movements upon binding of carbohydrate inhibitors in PPA. But a comparison of the KM and kcat values for starch degradation obtained from both a-amylases under optimal conditions shows that they are not signi®cantly different (Levitzki & Steer, 1974; Buonocore et al., 1976a). The same was observed for the disaccharide substrate p-nitrophenyl-a-D-maltoside (Levitzki & Steer, 1974; Strobl et al., 1997). The loop at 342 to 361 (in PPA), which is present in mammalian a-amylases but truncated in all insect a-amylases, is structurally close to the ¯exible loop discussed above (Figure 7(b)). In uncomplexed PPA, there exists one hydrogen bond between this loop and the Ê ). ¯exible loop (Asp356 Od2-His305 Ne2: 2.6 A No major structural rearrangement was detected in this region upon binding of carbohydrate inhibitors (Qian et al., 1994; Machius et al., 1996). In contrast to this, the loop in PPA consisting of residues 140 to 148 also moves upon binding of V-1532. The r.m.s. main-chain and side-chain movements for this segment sum up to 0.8 and Ê , respectively. 2.5 A Structural studies of PPA in complex with the proteinaceous inhibitors tendamistat from Streptomyces tendae (Wiegand et al., 1995) and the a-amylase inhibitor from Phaseolus vulgaris (common bean; Bompard-Gilles et al., 1996) showed that the same three loop elements as discussed above are involved in the binding of these inhibitors and undergo major structural rearrangements as compared to free PPA (Machius et al., 1996;

Structure of Yellow Meal Worm -Amylase

625

Figure 7. (a) Stereoview of a superposition of the trace models of TMA (orange) and PPA (blue). The positions of the calcium and chloride ions are depicted by a yellow and a purple star, respectively. Loops in PPA involved in binding of proteinaceous inhibitors and absent in TMA are emphasized in black. The orientation of the molecules approximately corresponds to that in Figure 4. (b) Stereoview of a superposition of the ``glycine-rich'' loop and the loop at position 342 to 361 (in PPA), present in PPA (blue), but absent from TMA (orange). The side-chain of His305 (PPA) is depicted in dark blue.

Bompard-Gilles et al., 1996). In the literature, several proteinaceous inhibitors of insect a-amylases have been reported not to be ef®cient against mammalian a-amylases and vice versa (Shainkin & Birk, 1970; Silano et al., 1973, 1975; Buonocore et al., 1977; GutieÂrrez et al., 1990; Chagolla-LoÂpez et al., 1994; for a review, see Richardson, 1990). Since the presence and/or absence of the above-mentioned loops is the main structural difference between the a-amylases from mammals and from insects, we suggest that an inhibitor exclusively directed against mammalian a-amylases makes crucial contacts to these loops, while an inhibitor solely directed against the insect enzymes might, for instance, be sterically hindered from binding by at

least one of the loops. Consequently, an inhibitor active against enzymes from both sources should make only minor or no interactions with these areas. The majority of interactions of tendamistat and of the bean a-amylase inhibitor to PPA are actually made in other contact areas. Therefore, it is not surprising that both inhibitors are also active against beetle a-amylases (Schroeder et al., 1995; G.W., unpublished results). The puri®cation and crystallization of TMA gives us the opportunity to analyze speci®c plant inhibitor/insect a-amylase interactions on a structural level. A comparison with inhibitor/PPA complexes might elucidate differences in inhibitor binding to a-amylases from both mammals and

626 insects. Therefore, we have initiated crystallographic studies on TMA in complex with inhibitors from various sources.

Materials and Methods Data collection The puri®cation of TMA, its amino acid sequence (Pir2:S75702) and its crystallization have been described (Strobl et al., 1997). The crystals used for the determination of the three-dimensional TMA structure belong to space group P212121 (as determined by molecular replaÊ , b ˆ 93.46 A Ê, cement) with cell constants a ˆ 51.24 A Ê , and contain one molecule per asymmetric c ˆ 96.95 A unit. The tight packing of the molecules within the crysÊ 3/Da tal is re¯ected by a Matthews parameter of 2.24 A (corresponding to 45% (v/v) solvent content), explaining Ê resolthe high diffraction power up to more than 1.6 A ution with conventional CuKa radiation. X-ray diffraction data were recorded on an imaging plate detector (MAR Research, Hamburg, Germany) attached to a Rigaku-Denki rotating Cu-anode generator operated at 5.4 kW providing graphite-monochromatized CuKa radiation. Data were processed using the MOSFLM v. 5.23 program (Leslie, 1991) and routines from the CCP4 suite (CCP4, 1994). Data collection statistics are summarized in Table 1. Structure solution and refinement The three-dimensional structure was solved by molecular replacement using the coordinates of unliganded pig pancreatic a-amylase (Machius et al., 1996). The program AMoRe (Navaza, 1994) was applied for rotational and translational searches, resulting in P212121 as the correct space group. The best solution (115.0; 57.0, 46.0; 0.2825; 0.3174; 0.2867; a, b and g are given in Eulerian angles; x, y and z are fractional cell coordinates) had a correlation coef®cient of 41.8% and a crystallographic Ê R-factor of 47.5% for data between 12.0 and 5.0 A resolution (the second best solution had values of 54.3% for the R-factor and 18.5% for the correlation coef®cient) after rigid-body re®nement using ``®tting'' (Navaza, 1994). After positional re®nement by means of X-PLOR (BruÈnger, 1991), the calculated (2Fobs ÿ Fcalc) and (Fobs ÿ Fcalc) electron density maps permitted modeling of differing side-chains, as well as insertions and deletions of TMA as compared with the searching model. The modeling was performed on a Silicon Graphics workstation using the program TURBO-FRODO (Roussel & Cambilleau, 1989). Six cycles of manual rebuilding and least-squares reciprocal space re®nement (including positional and individual constrained temperature factor re®nement) with X-PLOR ®nally resulted in a model with a crystallographic R-factor of 17.7% for Ê resolution after anisotropic data between 7.0 and 1.64 A data correction using X-PLOR (BruÈnger, 1991; see Table 1). The r.m.s. deviations from standard values Ê for bond lengths and were determined to be 0.008 A 1.482 for angles. One calcium cation and one chloride anion could be assigned unequivocally based on the strong positive difference density and reasonable temperature actors after re®nement: 261 water molecules were additionally introduced at stereochemically reasonable positions.

Structure of Yellow Meal Worm -Amylase

The Ramachandran plot was calculated with PROCHECK (Laskowski et al., 1993). Pictures have been produced with the programs MOLMOL (Koradi et al., 1996) and TURBO-FRODO (Roussel & Cambilleau, 1989). The coordinates of TMA have been deposited with the Brookhaven Protein Data Bank, accession number 1JAE, and will be released with a delay of one year, but are available from the authors on request until that time.

Acknowledgements Discussions with H. Fischer and S. Tan are gratefully acknowledged. This project was supported by the Deutsche Forschungsgemeinschaft (GI 159/1-2) and the ETH ZuÈrich.

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Edited by I. A. Wilson (Received 30 September 1997; received in revised form 19 January 1998; accepted 19 January 1998)