Crystal Structure of Calcium-depletedBacillus licheniformisα-amylase at 2.2 Å Resolution

JMB—MS 361 Cust. Ref. No. CAM 430/94

[SGML] J. Mol. Biol. (1995) 246, 545–559

Crystal Structure of Calcium-depleted Bacillus licheniformis a-amylase at 2.2 Å Resolution M. Machius, G. Wiegand and R. Huber Max-Planck-Institut fu¨r Biochemie, D-85152 Planegg-Martinsried Germany

The three-dimensional structure of the calcium-free form of Bacillus licheniformis a-amylase (BLA) has been determined by multiple isomorphous replacement in a crystal of space group P43 21 2 (a = b = 119.6 Å, c = 85.4 Å). The structure was refined using restrained crystallographic refinement to an R-factor of 0.177 for 28,147 independent reflections with intensities FObs > 0 at 2.2 Å resolution, with root mean square deviations of 0.008 Å and 1.4° from ideal bond lengths and bond angles, respectively. The final model contains 469 residue, 237 water molecules, and one chloride ion. The segment between Trp182 and Asn192 could not be located in the electron density, nor could the N and C termini. Cleavage of the calcium-free form of BLA was observed after Glu189, due to a Glu-C endopeptidase present in trace amounts in the preparation. BLA did not crystallize without this cleavage under the conditions applied. BLA exhibits the characteristic overall topological fold observed for other a-amylases and related amylolytic enzymes: a central domain A containing an a/b-barrel with a large protrusion between b-strand 3 and a-helix 3 (domain B) and a C-terminal greek key motif (domain C). Unlike in the other enzymes, domain B possesses a b-sheet made up of six loosely connected, twisted b-strands forming a kind of a barrel with a large hole in the interior. Topological comparisons to TAKA-amylase, pig pancreatic a-amylase and cyclodextrin glycosyltransferase reveal a very high structural equivalence for large portions of the proteins and an exceptionally pronounced structural similarity for calcium binding, chloride binding and the active site. None of the theories proposed to explain the enhanced thermostability of BLA showed a satisfactory correlation with the three-dimensional structure. Instead, sequence comparisons to the less thermostable bacterial a-amylase from Bacillus amyloliquefaciens (BAA) indicate that some ionic interactions present in BLA, but which cannot be formed in BAA, might be responsible for the enhanced thermostability of BLA. Keywords: a-amylase; X-ray structure; Bacillus licheniformis; a/b-barrel; thermostability

Introduction a-Amylases (a-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) are a family of endo-amylases that catalyse the hydrolysis of a-D-(1,4) glycosidic linkages in starch components or related carbohydrates, releasing malto-oligosaccharides and gluAbbreviations used: AAA, Aspergillus niger a-amylase; ADA, N-(2-acetamido)-2-iminodiacetic acid; BAA, Bacillus amyloliquefaciens a-amylase; BLA; Bacillus licheniformis a-amylase; BStA, Bacillus stearothermophilus a-amylase; CGT, cyclodextrin glycosyltransferase; m.i.r., multiple isomorphous replacement; PPA, pig pancreatic a-amylase; TAA, TAKA-amylase. 0022–2836/95/090545–15 $08.00/0

cose in the a-anomeric form. They are widely distributed in microorganisms, plants, and higher organisms and show varying action patterns. a-Amylases (and related amylolytic enzymes like cyclodextrin glycosyltransferases and a-glucosidases) from different organisms exhibit similar three-dimensional structures despite differences in their primary structure, with an a/b-barrel as a central part (domain A), a greek key motif as a separate domain C and at least one additional domain. Substrate binding is localized to a cleft between the a/b-barrel and domain B, comprising several b strands of variable length, depending on the species. Also common is a requirement for calcium, which maintains the structural integrity (Vallee et al., 7 1995 Academic Press Limited

JMB—MS 361 546 1959; Vihinen & Ma¨ntsa¨la¨, 1989; Violet & Meunier, 1989). The amino acids involved in substrate binding, chloride binding, calcium binding and catalysis are highly conserved among the different enzymes (Matsuura et al., 1984; MacGregor & Svensson, 1989; Boel et al., 1990; Klein & Schulz, 1991; Jespersen et al., 1991; Swift et al., 1991; Kizaki et al., 1993; Qian et al., 1993; Larson et al., 1994; Kadziola et al., 1994; Wiegand, Epp & Huber, unpublished). Bacillus licheniformis contains at least two aamylases with different amino acid sequences and catalytic action patterns (Yuuki et al., 1985; Kim et al., 1992). One of these, BLA, consists of a single polypeptide chain of 483 amino acids (Stephens et al., 1984; Yuuki et al., 1985). Although the organism itself is mesophilic, BLA exhibits a temperature optimum of about 90°C, which is quite different to the highly homologous a-amylases from Bacillus stearothermophilus (BStA) and Bacillus amyloliquefaciens (BAA) with temperature optima of about 75 and 60°C, respectively (Vihinen & Ma¨nsta¨la¨, 1989). This feature has led to its wide use in alcohol, sugar, and brewing industries for the initial hydrolysis of starch to dextrins, which are then converted to glucose by glucoamylases. Furthermore, they are used in desizing of fabrics, in the baking industry, in the production of adhesives, pharmaceuticals, and detergents, in sewage treatment, and in animal feed (Vihinen & Ma¨nsta¨la¨, 1989). BLA, as a flour additive, is also believed to be an important cause of protein contact dermatitis in bakers and allergic reactions in consumers of bread products (Morren et al., 1993). The preparation of BLA crystals suitable for X-ray analysis has already been described in the literature. Suzuki et al. (1990) reported crystallization with sodium sulphate as precipitant and EDTA as additive. Although Lee et al. (1991) did not use EDTA, the use of 100 mM ADA as buffer is likely to act as chelator of calcium at this high concentration as well. The presence of chelators seems to be indispensable for obtaining BLA crystals. In this paper we describe the structure solution of calcium-depleted BLA using multiple isomorphous replacement (m.i.r.) techniques, and ˚ resolution. This subsequent refinement to 2.2 A first reported structure of a microbial a-amylase, together with the available structures of a-amylases from Aspergillus oryzae (Swift et al., 1991), barley (Kadziola et al., 1994), and pig pancreas (Qian et al., 1993; Larson et al., 1994; Wiegand, Epp & Huber, unpublished) gives a line of phylogenetically distant a-amylases that should provide insights into the evolutionary processes regarding structure/function relationships and the basis of thermostability of some a-amylases. The BLA structure could also be used in protein design to adjust enzymatic and stability properties to industrial needs, and to localize and perhaps alter regions responsible for allergenic reactions in man.

Crystal Structure of Bacillus licheniformis a-amylase

Results Protein purification and crystallization As judged by native and SDS-PAGE, the crude extract provided by Sigma contains at least four stable isozymes with corresponding pI values in the range of 5 to 6 (data not shown). The most basic isozyme could be isolated in one step using ion exchange chromatography (data not shown). Analysis of BLA crystals grown in the presence of citrate and EDTA as well as in the presence of ADA, but without EDTA (Lee et al., 1991) using SDS-PAGE revealed that the protein was cleaved. N-terminal sequencing yielded the sequence DGNYDYLMY in addition to the native N terminus. Cleavage had taken place between Glu189 and Asn190, which deamidated to Asp190. This lead to the assumption that a Glu-C endopeptidase is present in the preparation, which could be confirmed by enzymatic tests. It is known that B. licheniformis, together with BLA and other enzymes, also releases a Glu-C endopeptidase into the culture medium (Kakudo et al., 1992; Svendsen & Breddam, 1992). Calcium is required to maintain the structural integrity of a-amylases (Vallee et al., 1959). Removal of calcium leads to decreased thermostability and/or decreased enzymatic activity (Violet & Meunier, 1989), or increased susceptibility to proteolytic degradation, as in our case. So far, it has not been possible to obtain crystals of BLA with bound calcium. Quality of the structure The final model of BLA resulted in an R-factor of ˚ and 0.251 0.177 in the resolution range of 8.0 to 2.2 A ˚ . It shows good geometry, between 2.3 and 2.2 A ˚ and with root mean square deviations of 0.008 A 1.4° from ideal bond lengths and bond angles, respectively. A Ramachandran plot (Ramachandran & Sasisekharan, 1968) of the f/c angles shows that 86.7% of the residues are in the most favoured regions, and only Tyr150, which is well defined in the density, is in the disallowed region as determined by the programme PROCHECK (Laskowski et al., 1992). A Luzzati (1952) plot gives an upper estimate ˚. of the error in the atomic positions of 0.2 A The presence of a chloride ion is supported by low ˚ 2 ) and temperature factors for the chloride ion (9.6 A the surrounding residues, which are well ordered. Furthermore this region shows a great structural homology to the chloride binding site in PPA. The (2Fobs − Fcalc ) maps contoured at 1s show continuous density for all main-chain atoms except the N-terminal Ala1 and Asn2, the C-terminal Arg483, and region 182 to 192 where cleavage had taken place. Several side-chains, all located on the protein surface, have quite high thermal parameters. These include Arg125, Asn126, Lys180, Ala181, Asp194, Arg437 and Arg442. There was no density for the side-chains of Ser310 and Glu465. The

JMB—MS 361 Crystal Structure of Bacillus licheniformis a-amylase

variation by residue number of the B-factors shows several mobile or flexible areas, which generally lie in loop regions and could only be built with iterative refinement. These include regions 120 to 133, 370 to 376, 413 to 422, 440 to 448, and 458 to 467. Description of the structure Figure 1a shows the overall folding of BLA with emphasis on the secondary structure elements as determined by the programme DSSP (Kabsch & Sander, 1983). Residues involved in helices and b strands are listed in Table 1. The overall topological fold of BLA is that observed for the other a-amylases: the central part (domain A, Ala101 to Ile103 and His205 to Tyr396) comprises an a/b or TIM barrel with a prominent excursion (domain B, Asn104 to Asp204) between b strand 3 (Ab3) and a-helix 3 (Aa3). The C terminus (domain C, Gly397 to Arg483) folds into a greek key motif. Details of the structure are discussed in the following sections. Structural comparisons

Topological alignment In order to deduce structural and functional properties common to the class of a-amylases and related amylolytic enzymes, a topological alignment with BLA, PPA, TAA and CGT has been performed through a least-squares fit of the b strands in the barrels. From visual inspection and sequence comparisons it was clear that b strand 1 (Ab1) of one protein structurally corresponds to the b strands 1 of the other proteins, Ab2 corresponds to the other Ab2s, and so on. Therefore no cyclic permutation of the b strand numbering, as suggested by Lesk et al. (1989) was necessary to align the barrels. The alignment of the barrel axes was determined from conserved residues. Figure 1 shows a cartoon of the four proteins after the fit. The common domains A, B and C all are located in similar regions of space. Not only the barrels, but also some b strands in the domains B and C could be recognized as structurally equivalent. To further refine the topological alignment, domains B and C were least-squares fitted using these equivalent b strands. The least squares fits were performed pairwise with routines built into O. Matching regions were recognized when the Ca atoms of at least three consecutive residues of one ˚ to the Ca atoms of at least protein were closer than 1 A three consecutive residues in another protein. These very rigid conditions resulted in a scaffold of structurally highly conserved regions. This scaffold ˚ distance cutoff in was filled by relaxing the 1 A ˚ ˚ increments of 1 A to a 4 A cutoff for the last step. Figure 2 shows the structurally equivalent residues in BLA, PPA, TAA and CGT. This alignment is in portions different to the sequence alignment of Holm et al. (1990), especially in domain B, which generally is the region with the lowest homology. Another amylolytic enzyme for which co-ordi-

547 nates are available, the a-amylase from Aspergillus niger (AAA), has not been included in the topological alignment because the structure is superimposable to TAA from the first to the last amino acid. The results of the topological alignment are the same as for TAA (data not shown).

Domain A Domain A is characterized by the a/b-barrel and complex loop structures connecting the strands and helices of the barrel. The barrel b-strands of the four proteins studied are remarkably similar with respect to length, sequence and topological location. Strands Ab1 and Ab2 have middle range lengths of three to five residues, strands Ab3, Ab4 and Ab5 have identical lengths in all four proteins with six residues for Ab3 and four residues for Ab4 and Ab5. Strands Ab6 and Ab7 generally are the shortest with two to four residues, whereas Ab8 is made up of five residues, except for PPA (eight residues, which is the longest strand at all). Similar but less strict rules hold for the barrel helices. Whereas less homogenous in length, Aa3 generally is the longest (16 to 18 residues), Aa5 is the shortest (nine to ten residues; PPA contains a four-residue 310-helix instead of an a-helix). Aa6 generally contains 11 residues. An interesting observation in BLA is the presence of proline within Aa7 and Aa8. As a consequence Aa7 is split into two parts; one of them is a 310-helix. In contrast, the proline in Aa8, although not able to form the characteristic hydrogen bonds, does not disturb the helix geometry. The barrel symmetry is broken in all four proteins by additional structures between the barrel helices and strands to different extents. Additional secondary structures common to all four proteins are two 310-helical segments (b-turns III, respectively) between Ab4 and Aa4, and before Aa7. The loop regions connecting the barrel strands and helices, normally the regions with the greatest structural diversity in a/b-barrel proteins, are topologically very similar in the four studied proteins. CGT and TAA show the highest similarity. The loop regions on the C-terminal side of the barrel (connecting a-helices with b-strands) generally are more complex than those of the N-terminal side and contain the active site, the calcium, and the chloride binding site.

Domain B Domain B, inserted between Ab3 and Aa3, is the least similar region of the four proteins studied. Although large portions are topologically equivalent, domain B of BLA exhibits features clearly different from domain B of the other proteins (Figure 1). As is typical for Bacillus a-amylases, this domain is much more complex. Six b-strands form a loosely connected and twisted antiparallel b-sheet. From this sheet two larger two-stranded sheets (made up of Bb3 and Bb4 as well as Bb5 and Bb6) fold back onto the large sheet and form a kind of barrel

JMB—MS 361 548

Crystal Structure of Bacillus licheniformis a-amylase

Figure 1. Comparative view of the overall topology of BLA (a), PPA (b), TAA (c) and CGT (d), emphasizing the secondary structure elements. Left side: view from the side of the barrel. Right side: view from above the barrel; b-strands are drawn as arrows, a-helices as spirals. Domain A is held in red, domain B in green, and domain C in light blue. Domain D and E in CGT are omitted for clarity. For PPA, the inhibitor Tendamistat (Wiegand, Epp & Huber, unpublished) is not shown. Chloride ions are indicated in yellow, calcium ions in cyan. All drawings of the structures were made with the programme MOLSCRIPT (Kraulis, 1991).

JMB—MS 361 Crystal Structure of Bacillus licheniformis a-amylase

Table 1 Secondary structure elements of BLA Domain A Ab1 Aa1 Ab2 Aa2 Ab3 Aa3 Ab4 A310 1 Aa4 Ab5 Aa5 Ab6 Aa6 A310 2 a A310 3 Ab7 A310 4 Aa7 Ab8 Aa7 Aa8

Leu7–Gln9 His21–Glu34 Ala39–Trp41 Lys80–Ser92 Asn96–Val101 Pro206–Leu223 Gly227–Leu230 Val233–His235 Phe238–Thr252 Phe257–Ala260 Leu267–Lys276 Ser282–Phe284 Val286–Ser296 Met304–Leu308 Val312–Lys315 Pro317–Lys319 Ala320–Phe323 Thr341–Phe343 Lys344–Thr353 Tyr358–Phe362 Tyr363–Tyr367 Lys381–Gln393

Domain B Bb1 Bb2 Bb3 B310 1 Bb4 Bb5 B310 2 Bb6

His105–Lys106 Ala111–Glu119 His133–His140 Trp157–His159 Phe160–Asp166 Leu171–Phe177 Asp194–Met197 Ala199–Ile201 Domain C

Cb1 Cb2 Cb3 Cb4 C310 Cb5 Cb6 Cb7 Cb8

Gln399–Tyr402 Ile408–Arg413 Leu424–Thr429 Gly434–Tyr439 Arg442–Asn444 Thr448–Asp451 Val460–Val461 Trp467–Val472 Val477–Val481

The assignment of the secondary structure elements was done with DSSP (Kabsch & Sander, 1983).

with a large hole in the interior. Two extrusions containing 310-helices are located between Bb6 and Bb3 and between Bb4 and Bb2. The region that is not visible in the electron density map is between Bb4 and Bb2. Bb1 (the entrance into domain B) and Bb6 (the exit from domain B) form a two stranded sheet that is also involved in the large sheet, and so define domain B as an entity which therefore might be a folding unit. The hydrogen bonding pattern of domain B of BLA is shown in Figure 3. The B domains of the other proteins also form b-sheets, but less complex ones. Common to these proteins is a six-residue helical segment, which in turn is not present in BLA.

Domain C Domain C in all four proteins includes a so-called greek key motif (Richardson, 1981) and forms a distinct globular unit. The overall topology is very similar and is only disturbed by a 310-helix after Cb4 in BLA, and two short b-strands after Cb5 and a 310-helical segment after Cb8 in PPA. As for the other structures, the final electron density map gives no hints for an attached carbohydrate moiety in BLA. There is still no distinct function that can be ascribed to this domain with certainty.

The active site, carbohydrate and inhibitor binding The catalytically active residues were already proposed for TAA and PPA in earlier works (Matsuura et al., 1984; Buisson et al., 1987; Quian et al., 1993). These are two aspartic acid and one glutamic acid and are located on the C-terminal side of the central b-barrel. After sequence alignment and

549 mutagenesis studies, corresponding residues could be defined for other carbohydrate converting enzymes that share the same catalytic mechanism (Klein & Schulz, 1991; Kadziola et al., 1994; Kizaki et al., 1993; Juncosa et al., 1994; Holm et al., 1990). These are listed in Table 2 for the four proteins studied and indicated in Figure 2. Figure 4 shows a superposition of the active site regions. Not only the three catalytically active residues are conserved, but also a number of residues in the vicinity. These residues are believed to play important roles in substrate binding. The structures of PPA in complex with cyclodextrins (Larson et al., 1994), a carbohydrate inhibitor (Qian et al., 1994), and with the protein inhibitor Tendamistat (Wiegand, Epp & Huber, unpublished) reveal that the ligands make strong hydrophobic interactions with the protein (together with a few specific hydrogen bonds) which therefore should also be expected for the natural substrate. These regions are not strictly conserved among the four proteins studied. The similarity is fairly high between PPA and TAA, much less distinct between PPA and CGT, and very poor between PPA and BLA, except for those residues that also are involved in calcium or chloride binding. This is due mostly to the clearly different B domain of BLA.

The calcium binding site A superposition of the residues involved in calcium binding is shown in Figure 5 and listed in Table 2. The calcium ion is not present in the BLA structure due to the crystallization conditions. Instead, there is a water molecule. The calcium binding site is located between the C terminus of the central b-barrel and domain B and serves as a link between the two domains. The architecture of the calcium binding site is well conserved and can be regarded as a distorted pentagonal bipyramid as described by Boel et al. (1990) for Aspergillus niger a-amylase. Generally, four residues serve as ligands. Three of them are strictly conserved: an asparagine in the loop region between domain A and B, an aspartic acid at the end of domain B, and the carbonyl oxygen atom of the histidine in the highly conserved region from the beginning of Ab4 to the beginning of Aa4. The fourth residue is located in a region of domain B, which forms an a-helix in PPA, TAA and CGT. The corresponding region is not visible in the BLA structure. Three water molecules complete the co-ordination sphere. Under the conditions used, BLA crystallizes only in its calcium-free and cleaved form. This suggests that there are specific crystal contacts around the putative calcium binding site that can form only after cleavage. There are ten direct crystal contacts in the ˚ (Table 3), five of BLA crystal structure less than 3.5 A which are ionic interactions. Apart from two hydrogen bonds, all interactions are between the B domains of crystallographically related molecules. Taken together with the fact that BLA crystals, upon addition of calcium, lose their ability to diffract X-rays, it can be concluded that conformational

JMB—MS 361 550

Crystal Structure of Bacillus licheniformis a-amylase

Figure 2. Topological alignment of BLA, TAA, PPA and CGT. Only structurally equivalent regions according to Ca positions are shown (see the text for details). a-Helices are drawn in red, b strands in blue, and 310-helices in yellow. Also indicated are the residues involved in calcium and chloride binding, and the catalytically active residues. For CGT and TAA, chloride ions have not been described by the authors. The calcium is not present in the BLA structure. (W) Unspecified number of structurally non-equivalent residues; ( ) 1 structurally non-equivalent residue.

JMB—MS 361 Crystal Structure of Bacillus licheniformis a-amylase

551

Figure 3. A representation of all b structures in domain B of BLA.

changes take place on binding or removal of calcium.

The chloride binding site Chloride allosterically activates PPA (Thoma et al., 1971; Yamamoto et al., 1988). A bound chloride ion can also be found in BLA (Figure 6). The binding site is located near the calcium binding site and the active site at the C-terminal end of the b-barrel. Its architecture is conserved to a similar degree as the calcium binding site and the active site. The chloride ion is co-ordinated by four ligands (NH2 of Arg229, ND2 of Asn326 and two water molecules). The co-ordination through the arginine and the asparagine is also found in PPA. In PPA there is an additional bidentate co-ordination through Arg337, replaced by a water molecule in BLA. A further water molecule is present in both structures, although it is not structurally equivalent. The residues involved in chloride binding and the distances are listed in Tables 2 and 4 and shown in Figure 6 together with the structures of the corresponding regions in CGT and TAA. The similarity to the chloride binding regions of the other proteins is clear, so that a chloride ion could be present in these structures as well, although the authors did not report it.

Ionic interactions Ionic interactions play an important role in stabilizing protein structures. The ionic interactions in BLA are listed in Table 5. Most of them connect loops to helices or stabilize helices or the barrel b strands. The only salt bridges at the interface between different domains are between Asp60 and Arg146 and between Asp204 and Lys237 (connecting domains A and B) and between Arg354 and Asp401 (connecting domains A and C). A few ionic interactions are conserved throughout BLA, CGT, PPA and TAA. All are located in the region around the active site (Table 5).

Other bacterial a-amylases and thermostability Since bacterial a-amylases, especially those from the genus Bacillus, are widely used in industry, and since the majority of industrial applications require their use at high temperatures (up to 110°C), much effort has been made to elucidate the molecular mechanisms of thermal inactivation of Bacillus a-amylases and the forces stabilizing their threedimensional structure, which is also of general interest. Much information about the thermal inactivation processes of the highly homologous a-amylases from Bacillus amyloliquefaciens (BAA),

JMB—MS 361 Crystal Structure of Bacillus licheniformis a-amylase

552 Table 2

Residues involved in structurally and functionally important features BLA

PPA

TAA

CGT

A. Residues at and around the active site Asp231 Asp328 Glu261 Gln9 Tyr56 Asp100 Val102 Lys234 Val259 Trp263 His327

Asp197 Asp300 Glu233 Phe13 Tyr82 Asp117 Val119 Lys209 Ile228 Leu232 His296

Asp206 Asp297 Glu230 His15 Tyr62 Asp96 Val98 Lys200 Phe231 Ile235 His299

Asp229 Asp328 Glu257 Gln19 Tyr100 Asp135 Ala137 Lys232 Phe255 Phe259 His327

Strictly conserved Strictly conserved Strictly conserved Not conserved Strictly conserved Strictly conserved Qualitatively conserved Strictly conserved Qualitatively conserved Qualitatively conserved Strictly conserved

Asn139 Asp199 His233 Ile190

Strictly conserved Strictly conserved Strictly conserved Not conserved

Arg227 Asn326 Ala356

Strictly conserved Strictly conserved Not conserved

B. Residues involved in calcium binding† Asn104 Asp200 His235 ‡

Asn100 Asp167 His201 Arg158

Asn121 Asp175 His210 Gln162

C. Residues involved in chloride binding§ Arg229 Asn326 Gln300>

Arg195 Asn298 Arg337

Arg204 Asn295 Ile326

† Calcium is not present in the BLA structure. The residues listed are topologically equivalent to the residues involved in calcium binding in the other proteins. ‡ The corresponding residue in BLA is not visible in the electron density. § For CGT and TAA the authors did not report the finding of chloride ions. The residues listed are topologically equivalent to the residues involved in chloride binding in BLA and PPA. > This residue is structurally equivalent to Arg337 in PPA but not involved in chloride binding.

Bacillus stearothermophilus (BStA), and Bacillus licheniformis (BLA) has been gathered (Tomazic & Klibanov, 1988a,b; Suzuki et al., 1989; Violet & Meunier, 1989; Declerck et al., 1990; Morand & Biellmann, 1991; Vihinen et al., 1990; Brosnan et al., 1992; Janecek & Balaz, 1992; Janecek, 1993). The half

lives of thermoinactivation at 90°C and pH 6.5 greatly increase in the series from BAA to BStA to BLA by two orders of magnitude (Tomazic & Klibanov, 1988b). A sequence alignment of the three amylases (Yuuki et al., 1985) together with the secondary

Figure 4. Superposition of the residues in and around the active site in BLA (red), TAA (green), PPA (blue), and CGT (black). AspA is 231 in BLA, 197 in PPA, 206 in TAA, and 229 in CGT. AspB is 328 in BLA, 300 in PPA, 297 in TAA, and 328 in CGT (see Table 2).

JMB—MS 361 Crystal Structure of Bacillus licheniformis a-amylase

553

Figure 5. Superposition of the calcium binding site in TAA (green), PPA (blue), and CGT (black). The equivalent region in BLA (red) is also shown, although calcium is not present in BLA due to the crystallization conditions. Only BLA residues are labelled (see Table 2).

structure elements of BLA is given in Figure 7. The homology is greatest between the least thermostable BAA and the most thermostable BLA, BStA showing more alterations in sequence. Therefore most lines of experiments compare BAA with BLA. According to the alignment, the three-dimensional structures of BAA and BStA can be expected to be very similar to that of BLA. Several explanations for the different thermostabilities have been proposed. Tomazic & Klibanov (1988a,b) hypothesized that the increased thermostability of BLA is due to additional salt bridges involving Lys385 and Lys88 and/or Lys253. The structure shows that these lysines do not take part in ionic interactions at all (Table 5), thus ruling out this proposal. There are four ionic interactions in BLA that are not possible in BAA according to the sequence alignment (Table 5), but others might exist. By constructing various chimeric genes from the structural genes for B. amyloliquefaciens and B. licheniformis Suzuki et al. (1989) have identified two regions in BLA which they suggest are determinants of thermostability. Region I comprises Gln178 and region II is Glu255 to Leu270. By means of site-directed mutagenesis, they altered the BAA sequence according to their proposals (deletion of Arg176 and Gly177 in region I, substitution of Lys269 for alanine in region II), rendering BAA almost as thermostable as BLA with respect to irreversible inactivation. It was therefore concluded that changes in charged residues (deletion of arginine and substitution of lysine for alanine) and therefore an increase in hydrophobicity may enhance thermostability of BAA. Region I in BLA is a loop on the surface of domain B. This loop is enlarged in BAA by two

extra residues, which could cause increased mobility of this region and a decreased thermostability of the whole protein. The alanine in region II of BLA also lies on the surface and is completely solvent-accessible, which most probably holds for the lysine in BAA at the same position, as well. We could not detect special interactions in regions I and II of BLA that could lead to a markedly increased thermostability, but these proposals cannot be completely judged by our study, because of the lack of the three-dimensional structures of BAA and of the mutants described above. Janecek (1993) assumed the b-strands of the a/b-barrel and helix Aa7 (which is completely hydrophobic) to form the hydrophobic interior and all other barrel helices to form the hydrophilic exterior. By calculating the hydrophobicity of these two parts and comparing the a-amylases of B. stearothermophilus, B. amyloliquefaciens and B. subtilis he found a correlation with the thermostabilities of these enzymes, such that increased hydrophobicity of the interior and simultaneously increased hydrophilicity of the exterior of the a/b-barrels were observed for more thermostable a-amylases. This study used predicted rather than experimentally determined secondary structure elements. Using the X-ray determined secondary structure elements of BLA and assuming that the location of the secondary structure elements in BAA and BStA is the same as in BLA, the correlation described above could not be reproduced for BAA, BStA and BLA (data not shown). None of the above mentioned theories is able to explain satisfactorily the enhanced thermostability of BLA. One cause could be the differences in the

Table 3 Intermolecular interactions Atom 1 D 121 OD1 D 121 OD2 R 127 NH1 E 132 OE1 E 132 OE1

Atom 2

Distance ˚) (A

Symmetry operator†

Atom 1

Atom 2

Distance ˚) (A

Symmetry operator†

K 180 NZ K 180 NZ K 180 NZ R 173 NH1 R 173 NH2

3.5 3.2 2.7 3.0 2.9

3 3 3 4 4

E 132 OE2 H 68 NE2 D 114 OD1 T 116 OG1 G 253 O

R 173 NH2 T 297 O Y 195 OH N 172 OD1 E 255 N

3.1 3.5 2.8 3.4 2.8

4 4 4 4 8

† Symmetry operations: 3, (1/2 − y, 1/2 + x, 3/4 + z); 4, (1/2 + y, 1/2 − x, 1/4 + z); 8, ( − y, − x, 1/2 − z).

JMB—MS 361 Crystal Structure of Bacillus licheniformis a-amylase

554

Figure 6. Superposition of the chloride binding site in BLA (red), and PPA (blue). The equivalent regions of CGT (black) and TAA (green) are also shown, although the authors did not report the finding of chloride ions. Only BLA residues are labelled (see Table 2).

number of ionic interactions in BLA and BAA (Table 5); further structural information on BAA is needed to substantiate this, however.

Materials and Methods Structural data Co-ordinates of TAKA-amylase and cyclodextrin glycolsyltransferase were taken from the Brookhaven Data Bank (Bernstein et al., 1977) with internal codes 6TAA and 1CGT, respectively. Co-ordinates of pig pancreatic a-amylase (in complex with Tendamistat) are a personal communication from Wiegand, Epp & Huber (unpublished). Enzyme preparation and crystallization BLA (purchased from Sigma as a crude extract) was extensively dialysed against buffer A (5 mM Tris-HCl, 1 mM CaCl2 , pH 8.5), subjected to ion exchange chromatography using TSK-DEAE Sepharose (Merck) and eluted with a linear gradient of 0 to 500 mM NaCl in buffer A. Size exclusion chromatography with Superdex G75 (Pharmacia) was sometimes also used to remove unwanted buffer substances. Prior to crystallization, BLA was dialysed against 0.4 M sodium citrate, 2.5 mM EDTA (pH 8.2) and concentrated to about 15 mg/ml. Crystals were grown by vapour diffusion at room temperature in droplets of 15 ml protein solution. The reservoir contained 10 ml of 0.60 to 0.69 M sodium citrate, 2.5 mM EDTA (pH 8.2). Crystals appeared after a few days and grew to a

maximum size of 1.2 mm × 0.3 mm × 0.3 mm within 2–3 months. They were harvested in 1 M sodium citrate, 2.5 mM EDTA (pH 8.2). The crystals belong to space group ˚ , c = 85.4 A ˚, P43 21 2 with cell constants a = b = 119.6 A a = b = g = 90°, and contain one molecule in the asymmetric unit. Crystals obtained with the procedure of Lee et al. (1991) showed the same properties as the crystals grown with citrate as precipitant. Testing for Glu-C endopeptidase activity 80 ml of sample solution were added to 900 ml buffer (50 mM Tris-HCl, 2 mM CaCl2 , pH 7.8) and the reaction started with 20 ml substrate solution (10 mM benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Boehringer, Mannheim, Germany) in dimethylformamide). An increase in UV-absorption at 410 nm corresponds to a Glu-C endopeptidase activity. To confirm the presence of a Glu-C endopeptidase, the sample was treated with the serine protease inhibitor diisopropylfluorophosphate (final concentration 250 mM) for 30 minutes, after which no activity could be observed any longer. N-terminal sequencing of BLA fragments 30 BLA crystals (about 1 mg protein) were dissolved in 6 M deionized urea (in 50 mM Tris-HCl, pH 8.0) and subjected to molecular sieve chromatography on a Superose 12 (Pharmacia) column. The two fragments were transferred to a ProBlotttmPVDF membrane (Applied Biosystems, Foster City, U.S.A.), washed with water and

Table 4 Co-ordination of the chloride ion Donor

BLA Acceptor

˚) Distance (A

Arg229 NH2 Asn326 ND2

−

Cl Cl −

3.1 3.2

Wat96 OH2 Wat63 OH2

Cl − Cl −

3.0 2.7

Donor Arg195 NH1 Asn298 ND2 Arg337 NH1 Arg337 NH2 Wat32 OH2

PPA Acceptor −

Cl Cl − Cl − Cl − Cl −

˚) Distance (A 2.9 3.2 3.4 2.8 3.5

Co-ordinating atoms which are structurally conserved in BLA and PPA are on one line.

JMB—MS 361 Crystal Structure of Bacillus licheniformis a-amylase

555

Table 5 ˚ between charged atoms) Ionic interactions in BLA (<3.5 A Atom 1 Asp18-OD1 Asp18-OD1 Asp18-OD2 Lys23-NZ Lys47-NZ Lys47-NZ Asp60-OD1 Asp60-OD2 Glu66-OE2 Lys80-NZ His91-NE2 Asp100-OD2 Glu113-OE1 Asp114-OD2 Asp121-OD1 Arg125-NH2 Asp166-OD1 Asp166-OD1 Glu167-OE1 Asp204-OD2 Arg229-NH1 Arg229-NH2 Arg242-NH1 Glu250-OE1 Glu271-OE1 Glu271-OE2 Asp303-OD1 Asp303-OD2 Asp303-OD2 Asp325-OD1 Glu336-OE1 Lys344-NZ Arg354-NH2 Arg354-NH2 Lys381-NZ His400-NE2 Asp404-OD1 His406-ND1 Arg456-NH1

Atom 2

˚) Distance (A

Involved domains

BAA†

BStA†

Arg24-NH1 Arg24-NH2 Arg24-NH2 Glu82-OE1 Asp63-OD1 Asp63-OD2 Arg146-NH1 Arg146-NH1 Lys80-NZ Glu222-OE2 Asp226-OD1 Arg229-NH1 His156-ND1 Lys136-NZ Arg127-NH1 Asp164-OD2 Arg169-NE Arg169-NH1 Lys170-NZ Lys237-NZ Asp231-OD2 Glu261-OE2 Asp243-OD2 Lys251-NZ Lys315-NZ His316-NE2 Arg305-NH2 Arg305-NE Arg305-NH2 Lys344-NZ Arg375-NH2 Asp365-OD1 Asp401-OD1 Asp401-OD2 Glu385-OE2 Glu414-OE1 Lys436-NZ Asp407-OD2 Glu458-OE1

3.1 3.2 2.8 2.8 2.8 3.3 2.7 2.8 2.9 2.9 2.9 3.2 2.8 3.0 3.1 3.3 2.8 3.1 3.0 3.0 3.0 3.4 2.6 3.1 3.5 3.3 2.9 2.9 3.5 2.9 2.7 2.8 2.7 3.2 3.1 2.7 2.9 3.2 3.1

A-A A-A A-A A-A A-A A-A A-B A-B A-A A-A A-A‡ A-A§ B-B B-B B-B B-B B-B B-B B-B B-A A-A§ A-A§ A-A A-A A-A A-A A-A A-A A-A A-A A-A A-A A-C A-C A-A C-C C-C C-C C-C

+ + + + + + + + + + + + + +

− − − + + + + + − + + −

+ + + + + + + +

+ + + + + + +

− + + + + − − + + + +

−

+

+

+

−

−

+ + + −

+ , Ionic interaction possible according to sequence alignment; − , sequence different from that of BLA, but ionic interaction might be possible. † The assignment of possible ionic interactions in BAA and BStA is made according to the sequence alignment shown in Figure 7. ‡ This ionic interaction is also present in CGT. § This ionic interaction is also present in PPA, TAA, and CGT.

N-terminal sequenced by Edman degradation using Applied Biosystems sequencers 470A and 473A according to the manufacturer’s instructions. Heavy atom derivative search Isomorphous derivatives were identified by conventional trial and error procedures. Crystals were transferred to heavy atom solutions, and two hours to 21 days later X-ray diffraction photographs were recorded on Buerger precession cameras. Heavy-atom derivative candidates were identified by visual comparison of native with complexed crystal photographs.

anode generator (Rigaku, Tokyo, Japan), apparent focal spot size 0.3 mm × 0.3 mm, 5.4 kW was used. Data sets were obtained by rotating the crystal about c* parallel to the spindel axis for 50° with a rotation range of the images of 1.0°. Reflections were evaluated using the programme MOSFLM (Leslie, 1991), and scaled and merged with the CCP4 package (Evans, 1991). Independent reflections with corresponding Friedel pairs were then further processed with the programme package PROTEIN (Steigemann, 1974). The data collection statistics for the native and the derivative data sets are summarized in Table 6. Phase calculation and structure solution

Data collection and processing X-ray intensity data were collected on Vaxstation 3100 controlled Hendricks/Lentfer X-ray image-plate systems (MarResearch, Hamburg, Germany). The crystals were cooled by a stream of cold air to about 2°C. Graphite monochromatized CuKa radiation from a RU200 rotating

The interpretation of heavy-atom derivatives and phase calculation was done with PROTEIN. One lead and two mercury positions were easily found from the interpretation of the difference Patterson maps. The resulting phases were used in difference and cross difference Fourier maps for the location of the other derivative heavy-atom sites.

JMB—MS 361 Crystal Structure of Bacillus licheniformis a-amylase

556

Figure 7. Sequence alignment of BLA, BAA, and BStA after Yuuki et al. (1985), and secondary structure elements of BLA (colouring scheme as in Figure 2). Only differences in the sequence of BAA and BStA compared to BLA are shown. Only BLA residues are numbered.

Heavy-atom parameters were refined against centric reflections only. Then all reflections were used to optimize the lack of closure and to calculate phases. Anomalous data were included to establish the enantiomeric space group with the programme SIGAT of the PROTEIN package. Of 70 trials six derivatives were finally used to solve the structure. The final atomic parameters of heavy atoms are given in Table 7.

Model building and refinement ˚ The m.i.r. phases with figure of merit = 0.62 at 3.0 A were further improved by solvent flattening using a solvent content of 45% (PROTEIN). A minimap was used to define molecular boundaries. Model building was accomplished with the programme packages FRODO (Jones, 1978), O (Jones et al., 1991), and MAIN (Turk, 1992), refinement was

Table 6 Data statistics Number of measurements

Number of reflections

Completeness (%)

Rsym † (%)

2.5 2.2

45207 52604

20115 28147

90.3 93.1

6.1 4.5

B. Derivative datasets‡ ETHG 3.0 PTPY 3.0 PBME 3.1 AUSO 3.0 PTCN 2.5 HGGL 2.5

22693 22212 20601 23627 38065 38737

11895 11791 11010 12175 20735 20572

91.3 90.5 93.1 93.6 93.3 92.7

7.4 8.9 12.0 4.9 12.5 9.1

Data set

Resolution limit ˚) (A

A. Native datasets NATI1 NATI2

† Rsym is defined as Si, hkl =I(i, hkl) − I(hkl)=/Si, hkl I(hkl), where i runs through the symmetry related reflections. ‡ ETHG, ethyl mercury chloride; PTPY, platinum (II)-(2,2'-6,2"-terpyridinium) chloride; PBME, methyl lead chloride; AUSO, gold sulphate; PTCN, potassium tetracyano platinate (II); HGGL, ethylal mercury thioglycolic acid.

JMB—MS 361 Crystal Structure of Bacillus licheniformis a-amylase

557

Table 7 Heavy atom derivative statistics Soaking conditions Fractional co-ordinates Data set ETHG

PTPY

Conc. (mM) 10

2.5

Time

Resol. ˚) (A

Rdev

F/E

x

y

z

21 d

3.0

18.2

1.38

2h

2.8

15.4

0.96

0.225 0.735 0.736 0.544 0.184 0.117 0.376 0.140 0.475 0.463 0.183 0.258 0.624

0.638 0.858 0.584 0.091 0.728 0.363 0.850 0.449 0.314 0.289 0.646 0.238 0.156

0.114 0.070 0.043 0.031 0.048 0.055 −0.008 0.096 0.070 0.102 0.095 0.071 0.055

PBME AUSO

100 10

21 d 2d

3.1 4.0

15.7 10.3

0.78 0.66

PTCN

10

1d

2.5

21.4

0.52

HGGL

10

1d

2.5

12.8

0.30

Nearest Rel. occ. amino acid 0.87 0.50 0.42 0.22 0.16 0.66 0.38 0.48 0.26 0.21 0.51 0.35 0.21

Dist. ˚) (A

His68 ND1 2.4 His68 NE2 1.5 Asp451 O 3.2 Glu113 OE2 2.7 Val339 CG 2.3 Lys234 O 4.0 His400 ND1 3.4 Asp200 OD2 3.1 Cluster between res. Thr217, ˚) Lys251, His247 (dist. 1 4.5 A His293 NE2 2.3 His316 ND1 1.1 His400 ND1 3.4

Abbreviations as for Table 6. Rdev : Mutual agreement factor between native and derivative data sets: Rdev = =FDS1 = − =FDS2 =/FDS2 . F/E: root mean square heavy atom contribution/residual.

done with XPLOR (Bru¨nger et al., 1987) using the parameter set of Engh & Huber (1991). Several b-strands and a-helices of the a/b-barrel could be located in the first electron density map. The TAA barrel was fitted to the emerging BLA barrel and used to identify other regions. A third of the a/b-barrel and the entire B domain could not be built at the first stage. The first model contained 328 residues (84 dummy alanines) in eight segments. It was subjected to a Powell minimization and gave an R-factor of ˚ . The resulting 0.456 in the resolution range of 8 to 3 A phases were combined with the m.i.r. phases and the model corrected as well as additional residues fitted into the electron density map. After three rounds of model building, Powell minimization, individual B-factor refinement, and phase combination, 468 residues could be ˚ . For identified and refined to an R-factor of 0.247 at 3.0 A subsequent rounds of model building and refinement only calculated phases were used. The resolution was increased ˚ . Adjustments of the model were in two steps to 2.5 A performed in (2Fobs − Fcalc ) and (Fobs − Fcalc ) density maps. Electron density maps obtained after simulated annealing were only used to identify problematic regions and ˚) side-chain orientations. At this stage (R-factor 0.247, 2.5 A a second native data set with higher resolution (NATI2; not merged with NATI1) was used for further refinement. Water molecules were automatically inserted using the programme package MAIN. Water molecules were accepted if the corresponding (Fobs − Fcalc ) density was at least 3.0s and geometric requirements for hydrogen bonding were fulfilled. A high density peak that would not account for a water molecule was interpreted as a chloride ion. When the (Fobs − Fcalc ) map showed no additional features, refinement was completed giving a BLA model consisting of 4032 protein (non-hydrogen) atoms within 469 residues, 237 water molecules and one chloride ion ˚ resolution. with an R-factor of 0.177 at 2.2 A

Acknowledgements The co-ordinates have been deposited with the Brookhaven Protein Data Bank and will be released with

a delay of two years, and are available from the authors on request in the meantime. This research was supported by grants from the Deutsche Forschungsgemeinschaft (Wi 1100/1). The authors acknowledge H.-C. Schneider who contributed significantly to the early stages of the work, Dr K.-H. Mann who has done the N-terminal sequencing, and Dr M. T. Stubbs for helpful discussions.

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Edited by I. A. Wilson (Received 19 September 1994; accepted 5 December 1994)