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Structural basis for the inhibition of mammalian and insect α-amylases by plant protein inhibitors
Biochimica et Biophysica Acta 1696 (2004) 171 – 180 www.bba-direct.com
Review
Structural basis for the inhibition of mammalian and insect a-amylases by plant protein inhibitors Francßoise Payan * Architecture et Fonction des Macromole´cules Biologiques, UMR6098, CNRS and Universities Aix-Marseille I and II, 31 Chemin Joseph Aiguier, F-13402 Marseilles, France Received 8 May 2003; accepted 23 October 2003
Abstract a-Amylases are ubiquitous proteins which play an important role in the carbohydrate metabolism of microorganisms, animals and plants. Living organisms use protein inhibitors as a major tool to regulate the glycolytic activity of a-amylases. Most of the inhibitors for which three-dimensional (3-D) structures are available are directed against mammalian and insect a-amylases, interacting with the active sites in a substrate-like manner. In this review, we discuss the detailed inhibitory mechanism of these enzymes in light of the recent determination of the 3-D structures of pig pancreatic, human pancreatic, and yellow mealworm a-amylases in complex with plant protein inhibitors. In most cases, the mechanism of inhibition occurs through the direct blockage of the active center at several subsites of the enzyme. Inhibitors exhibiting ‘‘dual’’ activity against mammalian and insect a-amylases establish contacts of the same type in alternative ways. D 2003 Elsevier B.V. All rights reserved. Keywords: a-Amylase; Inhibitor; Bean inhibitor; Cereal inhibitor; Wheat inhibitor; X-ray structure
1. Introduction a-Amylases (a-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) are a group of enzymes widely distributed in microorganisms, plants and animal secretions which catalyze the hydrolysis of the (a-1,4) glycosidic linkages in starch and various oligosaccharides. Carbohydrate normally constitutes the main component of the human diet and starch accounts for most of the ‘carbohydrate pool’. The cleavage of starch by a-amylases constitutes the first step in the enzymatic degradation of polysaccharides which is essential
Abbreviations: 0.28 and 0.19, a-amylase proteinaceous inhibitors from wheat kernel with molecular weights of 12,000 and 24,000 and gelelectrophoretic mobilities, relative to bromophenol blue, of 0.28 and 0.19, respectively; TMA, a-amylase from the yellow mealworm (larvae of Tenebrio molitor); HPA, human pancreatic a-amylase; HSA, human salivary a-amylase; PPA, pig pancreatic a-amylase; a-AI, a-amylase inhibitor from bean Phaseolus vulgaris; RBI, bifunctional a-amylase/ trypsin inhibitor from ragi (Eleusine coracana Gaertneri, Indian finger millet); AAI, a-amylase inhibitor from Amaranthus hypocondriacus seeds; r.m.s.d., root mean square deviation; PDB ID-code, Brookhaven Protein Data Bank accession reference * Tel.: +33-4-9116-4506; fax: +33-4-9116-4536. E-mail address: [email protected] (F. Payan). 1570-9639/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2003.10.012
in carbohydrate assimilation. Inhibition of a-amylases reduces the post-prandial glucose peaks, which is of particular importance in patients with diabetes. Carbohydrate in the form of starch also forms the bulk of the diet of insects such as the meal beetle Tenebrio molitor and its larvae, the yellow mealworm. These insects are cosmopolitan pests feeding on seed products. Plants have evolved defence strategies to counteract these effects through enzyme inhibitors impeding the action of insect gut digestive a-amylases and proteinases. Understanding the inhibitory mechanisms exerted on mammalian and insect a-amylases is a key step in the design of high affinity/selectivity a-amylase inhibitors having potentials in various fields, from the treatment of diabetes to crop protection. The number of proteinaceous a-amylases inhibitors isolated and identified so far is extremely large. They occur in microorganisms, higher plants and animals [1]. Proteinaceous a-amylases inhibitors can have different polypeptides scaffolds and can be grouped by their tertiary structures into six classes: lectin-like, knottin-like, cerealtype, Kunitz-like, g-purothionin-like and thaumatin-like. Plant a-amylase inhibitors, which are abundant in cereals and leguminosae, have been extensively studied. The area has benefited from the recent determination of many struc-
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tures of a-amylases, inhibitors and complexes: see Ref. [2] for a review describing the properties of insect a-amylases and their inhibitors. Three-dimensional (3-D) structures of plant protein inhibitors in complex with a-amylases involve a lectin-like a-amylase inhibitor from bean (Phaseolus vulgaris): (a-AI) [3– 5], a cereal-type a-amylase inhibitor: bifunctional a-amylase/trypsin inhibitor from ragi (Eleucine coracana Gaertneri; Indian Finger Millet) (RBI) [6], a knottin-type a-amylase inhibitor, amaranth a-amylase inhibitor (AAI) [7]; a Kunitz-like a-amylase inhibitor, barley a-amylase/subtilisin inhibitor (BASI) [8]. This review is aimed at displaying structural characteristics of the inhibition mechanism of mammalian and insect a-amylases by plant inhibitors by way of examples Refs. [3 –7] and also reports 3-D structure of an insect a-amylase in complex with the monomeric wheat inhibitor 0.28 (cereal-type). The mechanism of inhibition of the endogenous barley a-amylase 2 (AMY2) by BASI [8] is reported elsewhere in this issue.
2. Interaction of carbohydrate inhibitors with mammalian and insect A-amylases: structural basis for their catalytic mechanism Insights into the catalytic mechanism of mammalian aamylases came from the resolution of 3-D structures of PPA and human pancreatic a-amylase (HPA) in complex with carbohydrate inhibitors. Agents having inhibitory effects on PPA and on the sucrase and maltase activities of intestinal disaccharidases were first discovered in vitro in actinomycetes broths [9]. These substances correspond to homologous pseudo-oligosaccharide compounds (trestatin family), which are by now well-known. They contain a specific structural entity named acarviosine (an unsaturated cyclitol unit linked to a 4,6-dideoxy-4-amino-D-glucose) which is the essential structural unit responsible for the inhibitory function of this family of inhibitors. This unit is linked by a-(1,4)-O-glycosidic bonds to a variable number of glucose residues. One particular compound, the pseudotetrasaccharide acarbose, in which the acarviosine unit is linked to a maltose molecule [9], is a potent inhibitor of aamylases used to treat diabetic diseases. The architecture of mammalian and insect a-amylases consists of three domains: the catalytic core domain (A), comprising a (h/a)8 barrel, contains an extended loop inserted between the third h-strand and the third a-helix (called domain B). The C-terminal domain (domain C) forms a distinct globular unit forming a Greek key motif (Fig. 1). Both mammalian and insect enzymes require one essential calcium ion to maintain their structural integrity, and are activated by chloride ions. Elements from domain A and B are involved in the architecture of the three most functionally important sites: the active site, the calcium-binding site, and the chloride-binding site [10 –13].
Fig. 1. MOLSCRIPT [49] diagram of the a-amylase (PPA) structure. Domain A is shown in red, domain B in yellow and domain C in purple. The calcium ion (blue sphere) and the chloride ion (yellow sphere) are also shown in the immediate vicinity of the catalytic center. An acarbose-derived ligand (ball-and-stick representation in green) is bound at the active site cleft. Monosaccharide and disaccharide ligands (in ball-and-stick representation) are shown bound to the surface binding sites [50].
An atomic description of the interactions occurring within the active site depression between PPA and carbohydrate inhibitors have been published along with the refined structures of PPA – acarbose complexes [14,15] (determined using two different crystal forms), that of PPA complexed with V-1532, a member of the trestatin family [16] and that of HPA interacting with trestatin-derived compounds (acarbose and B4) [5,17]). The strong inhibition of a-amylases by these compounds is mainly attributed to the binding at subsite 1 (where the reaction intermediate is formed) of the cyclitol unit. In half-chair conformation it mimics the substrate distortion expected to occur in the transition state (for a review on possible reaction mechanisms for glycosidases, see Ref. [18]). The adjacent glycosidic bond is N-linked and prevents enzymatic hydrolysis; acid hydrolysis of acarbose leads to the breakage of both O-glycosidic bonds while enzymatic hydrolysis by a-amylase only results in the cleavage of the linkage between the two glucose units [19].
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More recently, the high-resolution structure of PPA complexed with the acarviosine-glucose member of the trestatin family was solved [20]. It was shown that an hexasaccharide species, filling subsites 4 though + 2 and composed of two a-1,4-linked original molecules remained bound to the enzyme active site (Fig. 2; PDB ID-code, 1HX0). These Xray structures clearly show a subset of residues in PPA active site directly involved in binding the inhibitor. The structural arrangement observed upon binding of the carbohydrate inhibitors indicates that PPA possesses all the requirements for hydrolysis to occur via the general acid hydrolysis mechanism. The widely recognized two-step mechanism originally proposed by Koshland [21] for retaining glycoside hydrolases requires the presence of two carboxyl-containing amino acids, the acid/base catalyst and the nucleophile, responsible for the formation of the glycosyl – enzyme intermediate [22]. The crystallographic data of PPA in complex with acarbose suggested that both Asp197 and Glu233 are required to produce the h-linked glycosyl – enzyme intermediate [14]. McCarter and Withers [23] later confirmed that Asp197 serves as the catalytic nucleophile. In addition, Xray crystallographic studies on a family 13 cyclodextrin glucanotransferase (CGTase) mutant (Glu257Gln), trapped as a glycosyl – enzyme intermediate via a reaction with 4deoxymaltotriosyl a-fluoride, showed the existence of an unequivocal h-glycosidic covalent bond located between the Asp229 Oy1 atom (corresponding to Asp197 in PPA) and the substrate anomeric C1 atom [24]. In the trestatin-derived
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PPA and HPA complexes, Glu233 and Asp300 are hydrogen-bonded to each other via an intervening water molecule, as well as to the nitrogen atom of the non-hydrolyzable Nglycosidic bond. Glu233, located in close proximity to the chloride ion and an intervening water molecule, is the most appropriate candidate for the role of general acid/base catalyst as previously substantiated by the results of structural analysis [14,25] and recently confirmed by Rydberg et al. [26]. Insect and mammalian a-amylases display high homology in their primary and tertiary structures. For example, a-amylase from the yellow mealworm (TMA) and porcine pancreatic a-amylase (PPA) share 54% sequence identity [27], and 410 structurally conserved Ca atoms super˚ [13]. Based on PPA imposed with an r.m.s.d. of 2.2 A crystallographic data [16], a model for the binding of a pseudo-hexasaccharide ligand in the active site depression of TMA has been constructed [6]. This model indicates that TMA can accommodate six saccharide units. Asp185, Glu222, and Asp287 (corresponding to Asp197, Glu233, and Asp300 in PPA) are proposed as key residues for catalysis [13]. It is likely that both mammalian and insect a-amylases have a similar mechanism of action. More recently, the X-ray structures of plant protein inhibitors in complex with either the pancreatic a-amylases HPA and PPA, or TMA have shed light on the structural basis for the inhibitory mechanisms of mammalian and insect a-amylases.
Fig. 2. Electron density of an acarbose-derived ligand bound to the 4 to + 2 subsites of PPA [20]. In the acarbose-derived extended ligand, the cyclitol unit is bound at subsite 1 (where the reaction intermediate state is formed); nomenclature n/ + n, assuming the cleavage to take place between subsites 1 and ˚ (at 1.38 A ˚ resolution). The figure was prepared using + 1, is according to Ref. [51]. The map shown is a 2Fo Fc electron density contoured at 0.5 10 3 A TURBO-FRODO [52].
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3. Interaction of plant proteinaceous inhibitors with mammalian and insect A-amylases—structural basis for the inhibition mechanism 3.1. The bean P. vulgaris a-amylase inhibitor The seeds of the common bean, P. vulgaris, contain a family of plant defense proteins including an a-amylase inhibitor, a-AI [28]. Two forms of the a-amylase inhibitor have been described, a-AI1 and a-AI2 which differ in both their primary sequence and their inhibitory activity towards bruchids [29 –31]. a-AI1 is a plant defence protein and a potent inhibitor of both mammalian and insect a-amylases (for PPA, Kd = 3.5 10 11 M at neutral pH) [32]. The overall architecture of the bean-inhibitor corresponds to a classical lectin fold [3] (PDB ID-code, 1DHK). The X-ray structures of the inhibitor in complex with either HPA, PPA, or TMA have been determined [3 –5]. 3.1.1. Interaction of a-AI1 with the mammalian a-amylases HPA and PPA The main interactions with PPA and HPA occur directly at the V-shaped depression of the a-amylase active site, which is also the binding site for acarbose [14]. The inhibition process is very similar for both enzymes. In both a-AI1 –HPA and aAI1 – PPA complexes, two facing hairpin loops emerging from the h-sheet fold of the inhibitor lie fully in the active site depression forming extensive hydrogen-bonding, hydrophobic and water-bridged contacts with the active site residues of the enzyme. At the heart of the substrate-binding site, two tyrosine residues (Tyr37 and Tyr186) protruding from the extremity of the two hairpin loops of a-AI combine to provide interactions with the catalytic residues, nucleophile and acid catalyst of the enzyme (Asp197 and Glu233, respectively). The interactions occurring in the region of subsites 1, + 1, + 2 are highly conserved in the complexes between carbohydrate or proteinaceous inhibitors and pancreatic a-amylases (PPA and HPA). Hydrophobic interactions also occur between the substrate’s surface and the hydrophobic residues lining the entrance of the cleft (subsite 2, 3). These interactions are followed by protein – protein interactions involving areas further away from the catalytic center, namely the loop 303 – 312, the loop at position 237 –240, the loop 347 – 357, and the loop 140 – 150 from domain B. Although the mechanisms of inhibition of HPA and PPA by a-AI are very similar, a close inspection of the structure of the a-AI1– HPA and a-AI1– PPA complexes showed the presence of additional hydrogen bonds between the inhibitor and domain B of the human enzyme. PPA and HPA show discrepancy in their amino acid sequences in this area which are also believed to influence the substrate specificity of the two enzymes [5]. A different network of interactions was also observed in the loop regions 303 – 312 and 347– 357 of domain A.
The structural changes induced in response to the binding of a-AI at the active site of the enzyme are very different from those induced by the carbohydrate inhibitor acarbose. The main displacement occurring upon the binding of the carbohydrate inhibitors involves the ‘‘flexible loop’’ (residues 303 – 309) [14]. An additional area that undergoes significant conformational changes is the loop extending from residues 237 –240, which forms the surface edge at the substrate-reducing end periphery of the active site depression. Residues from this loop and from the ‘‘flexible loop’’ are involved in the architecture of subsite + 2. Also affected by the carbohydrate inhibitor binding process is the loop segment at position 140 –150 of the domain B [5,15,20]. Two particular acarbose-binding residues, the catalytic residue Asp300 and residue His305 undergo ˚ takes place substantial changes. A movement of about 5 A at residue His305 which approaches the acarbose-derived ligand from the solvent side and forms a strong hydrogen bond with the residue in subsite 2. The side-chain of the catalytic residue Asp300 undergoes an induced conformational change upon substrate binding. It rotates about 60j around the Ca –Ch bond and becomes hydrogen bonded to the acid catalyst Glu233 via an intervening water molecule. Upon substrate binding, the ‘‘flexible loop’’ moves in toward the saccharide, thus reducing the cleft breadth. In contrast, when complexed with a-AI, the same loop moves out toward the solvent, pushed away by the inhibitor as a result of the tight-binding inhibition process. This movement is accompanied by the readjustment of the surrounding regions; in particular, the loop region including residues 351 – 359 in domain A. The side chain of the catalytic residue Asp300 shows the same orientation in both the free structure of PPA and the a-AI – PPA complex structure (instead of its substrate-induced conformation), suggesting that Asp300 adopts this functional position when a sugar unit is bound at subsite 1. In summary, the inhibitor a-AI completely blocks the substrate-reducing end of the enzyme cavity and prevents access to the other end via a steric hindrance process. The inhibitor triggers substrate ‘‘mimetic’’ interactions with the binding subsites on the enzyme and all the catalytically competent components of the enzyme are targeted (as an example, see Fig. 3a and b). 3.1.2. Interaction of a-AI1 with the insect a-amylase TMA Although TMA has the structure typical of a-amylases [13] (PDB ID-code, 1JAE) with an active site showing a V-shaped depression very similar to that of pancreatic aamylases, large deviations occur in the loops and particularly in the loop segments next to the active site region. However, structural analyses of the complex between aAI1 and TMA [4] (PDB ID-code, 1viw) indicated that the strong contacts occurring between the inhibitor and the catalytic cleft of the PPA, HPA and TMA enzymes are highly conserved. The two hairpin loops of the inhibitor
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residues Tyr186 and Tyr37, which project into the heart of the catalytic site and to the flanking residue Asp38, which occupies the subsite + 2 and blocks the access to the region of the ligand-reducing end. In summary, despite differences occurring in the interacting loops of the enzymes, a-AI1 inhibits both the insect and mammalian a-amylases via the same inhibitory mechanism. The protein partners being able to produce the same type of contacts in alternative ways. 3.2. The ragi bifunctional inhibitor The bifunctional a-amylase/trypsin inhibitor RBI from ragi (E. coracana Gaertneri; Indian finger millet) is the prototype of a cereal inhibitor superfamily. It is a monomer of 122 amino acids [33] with five disulfide bonds. It is a potent inhibitor of TMA (Ki = 15 nM) [6,34] and PPA (Ki = 11 nM for PPAII isoform; [34]). The 3-D structure of RBI was solved using nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography procedures [35,36]. The globular fold of RBI consists of four helices with a simple ‘‘up and down’’ topology and a small antiparallel h-sheet (PDB ID-code, 1B1U).
Fig. 3. (a) A trestatin-derived inhibitor bound to the active site of HPA. The five occupied subsites are labeled 3 to + 2. The catalytic residues, nucleophile and acid/base catalyst are all shown in red; model produced using GRASP [53]. (b) Selected residues of the proteinaceous inhibitor aAI interacting in the active site of HPA. The figure was adapted from Ref. [5]. Similar views of the TMA active site comparing interaction with: (a) a pseudo-hexasaccharide inhibitor V-1532 [16], and (b) RBI and AAI residues are available in Refs. [6,7], respectively.
(residues 29 –46 and 171 –189), which protrude into the active site of TMA, establish a similar network of hydrogen bonds, with the residues of the substrate-docking region, to that observed in the complexes between pancreatic a-amylases and a-AI1. The catalytic residues of TMA are strongly hydrogen-bonded to the inhibitor
3.2.1. Interaction of RBI with the insect a-amylase TMA In the RBI – TMA complex (PDB ID-code, 1TMQ) [6], the inhibitor binds to the active site of the enzyme. As observed with the bean inhibitor a-AI1, RBI interacts with residues from domains A and B which line the substrate binding site of TMA. In the RBI –TMA complex, the subsites of TMA (defined by analogy with those of PPA) are completely blocked by RBI residues. Inhibition by RBI mainly requires the harrow-head-like segment composed of N-terminal residues Ser1 – Ala11 and residues Pro-52 – Cys-55, which protrude into the TMA substrate binding groove and directly target the catalytic residues [6]. It is worth noting that although residues 1 –5 are flexible in the solution structure of free RBI, they adopt a 310-helical conformation in the complex, filling the substrate binding site of TMA. As explained above, the binding of a-AI to PPA and to TMA occurs through very similar contacts to residues in and around the active site cleft. The interaction between the shortened loops (corresponding to ‘deleted’ regions) of TMA and a-AI being maintained via neighboring TMA residues. In view of these results, the inhibition strategies for two other proteinaceous inhibitors, RBI and Tendamistat (a proteinaceous inhibitor from Streptomyces) against PPA and TMA have been compared [6,7]. Models of RBI – PPA and of Tendamistat– TMA complexes were constructed on the basis of the X-ray structures of the complexes RBI – TMA [6] and Tendamistat – PPA [37], respectively. Structural comparisons of these models with the respective experimentally determined structures led to the conclusion, similar to that of Nahoum et al. [4], that all three proteinaceous inhibitors
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(RBI, a-AI and Tendamistat) can inhibit both mammalian and insect a-amylases using alternative ways [7]. 3.3. The amaranth a-amylase inhibitor AAI specifically inhibits a-amylases from insects, but not from mammalian sources [38]. With 32 amino acids, AAI is the smallest a-amylase inhibitor described so far. Based on its overall fold, its three-stranded twisted hsheet and the topology of its disulfide bonds, AAI has been classified as a knottin-like protein (for a detailed bibliography, see Refs [2,7]). 3.3.1. Interaction of AAI with the insect a-amylase TMA The crystal structure of TMA in complex with AAI ˚ resolution (PDB ID-code, 1CLV) was determined at 2.0 A [7]. The inhibitor binds into the active site depression of TMA, blocking the central four sugar-binding subsites. The structure of the AAI – TMA complex shows that inhibition, as for the lectin-like inhibitor (a-AI), occurs through blockage of the substrate-binding sites by residues from AAI targeting the active site residues of TMA [7]. In the interaction of AAI with TMA, only Asp287, one of the catalytic residues of TMA (corresponding to Asp300 in PPA) forms a salt bridge directly with the inhibitor residue Arg7. The other catalytic residues: the nucleophile and acid catalyst, Asp185 and Glu222, respectively (by analogy with PPA residues Asp197 and Glu233), are connected via an intricate water-mediated hydrogen-bonding network to the inhibitor residues. In contrast, in the inhibition scheme of TMA by a-AI and RBI, all three catalytic residues establish direct hydrogen bonds with the functional groups of inhibitor residues. The structural analysis of the AAI – TMA complex [7] provides insights into the lack of recognition of mammalian a-amylases [38]. In particular, the extended network of hydrophobic interactions observed in other complexes of a-amylases and proteinaceous inhibitors [3,6,37] could not be identified [7]. Comparison of the modeled AAI – PPA complex with the AAI – TMA complex X-ray structure identified six hydrogen bonds specific for the AAI – TMA complex and probably responsible for the high specificity of the AAI inhibitor against insect a-amylase. 3.4. The wheat inhibitors Wheat kernels are particularly richly endowed with aamylase inhibitors affecting both insect and mammalian enzymes. Some of these substances specifically inhibit insect enzymes, in that they inhibit a-amylases from insects strongly but inhibit mammalian a-amylases only weakly or not at all. A set of a-amylase inhibitors were purified from wheat flour: they display molecular masses of about 12, 24, and 60 kDa and are members of the cereal superfamily of a-amylase inhibitors. They act as monomers, homodimers or hetero-oliogomers.
The insect-specific a-amylase inhibitors are mostly monomeric proteins (members of the 12 kDa family); the members from the 24 kDa family actively inhibit both mammalian and insect a-amylases [39]. The T. molitor aamylase (TMA) is effectively inhibited by a number of water-soluble protein components of the wheat kernel, particularly those termed inhibitors 0.28 and 0.19, which refers to their gel electrophoretic mobility relative to bromophenol blue [39,40]. The inhibitor 0.28 is a monomer with a molecular weight of 12 kDa, whereas the inhibitor 0.19 is a dimer with a molecular weight of about 24 kDa [40]. The dissociation constants of the a-amylase inhibitor 0.19 and a-amylase inhibitor 0.28 complexes are 0.85 and 0.13 nM, respectively [41]. 3.4.1. The wheat inhibitor 0.19 The inhibitor referred to as 0.19 inhibits a-amylases from human saliva, pig pancreas, chick pancreas, the yellow mealworm, and Bacillus subtilis [42]. The molecule has 124 amino acid residues [43] and its amino acid sequence is 26% identical to that of RBI [33]. It is the only other member of the cereal family with a known 3-D structure (PDB ID-code, 1HSS) [44]. The crystallography study revealed that 0.19 is an homodimer in accordance with the previous results obtained by gel chromatography [40,45]. In the crystal structure, the asymmetric unit has four molecules of 0.19, each corresponding to a monomer of 124 amino acid residues. Each subunit consists of four major a-helices arranged in an ‘‘up and down’’ pattern and two short antiparallel h-strands [44]. The h-strands are included in a characteristic protruding hairpin loop inserted before a one-turn helix (helix-5) into the Cterminal region of the molecule. The electron density for two regions including residues 1– 4 and 68 – 78 was not present in all four subunits, suggesting that these residues have multiple conformational states [44]. The 3D structure of the 0.19 subunits appears to be very similar to that of RBI, although two characteristic loops differ markedly. Firstly, the protruding segment which occurs in RBI between helices a3 and a4 (including short antiparallel h-strands) is absent from the structure of 0.19 (Fig. 4) for which, most of the residues in this loop region are disordered (residues 68 –78). Secondly, the characteristic protruding loop present in 0.19 on the other side of the molecule (where the C-terminal segment is located) is more extensive than the corresponding loop in RBI. 3.4.2. Interaction of the wheat inhibitor 0.19 with mammalian and insect a-amylases In order to identify the factors determining the specificity of the wheat inhibitors acting on mammalian and insect a-amylases, the overall structural similarity between RBI and 0.19 was used to model the interaction of 0.19 with TMA and HSA [42]. Despite some steric clashes between a few side-chains from the two partners, 0.19 inhibitor could be readily accommodated in the binding
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3.4.3. The wheat inhibitor 0.28 The monomeric wheat a-amylase inhibitor known as 0.28 is one of the most fully characterized inhibitor of this kind. It is highly active against a-amylase of T. molitor (TMA) and only weakly inhibits the a-amylases from human saliva and pancreas and from the pancreas of some avians [39]. The 123 amino acids sequence [46,47] is 60% identical to that of 0.19 and 26% to that of RBI. All 10 cysteine residues in the molecule form disulfide bridges [48]. The 3-D structure of the complex between TMA and 0.28 has recently been solved (F. Payan, V. Nahoum, S. Strobl, R. Glockshuber, R. Huber, E. Poerio, unpublished results). Below are some of the structural characteristics of this complex.
Fig. 4. Interaction of the cereal inhibitors with the substrate binding cleft of TMA. (A, top) The molecular surface of TMA is shown in grey. The main chains of RBI, 0.28 and 0.19 are represented by grey, green, and red tubes, respectively. The free structure of 0.19 is superimposed onto the structure of 0.28 (as bound to TMA). The loop L4 is circled; the N-terminal segment is only present in the X-ray structures of RBI and 0.28 in complex with TMA (see text). (B, bottom) A close-up of the loops L4 as observed in 0.19 (in red) and 0.28 (in green), as bound to TMA, is shown. Both figures were made with SPOCK [54].
site of the enzymes in the same orientation as seen for RBI. At the C-terminus of 0.19, the two residues 123 – 124 superimposed with the facing enzyme region and were therefore deleted from the model. The inhibitor interface residues that differ between 0.19 and other wheat a-amylase inhibitors of this family (see Ref. [42] for a detailed nomenclature) were analyzed using the modeled complex of HSA-0.19. As a result, three inhibitor spots of interest were proposed. The first is residue His47, situated, in the model, near residue Glu349 of HSA; the second concerns Ser49 of 0.19, closely packed by Lys352, and Asp356 of the long loop of HSA; the third region of interest is the sequence Val – Val – Asp –Ala from residues 104 –107 of 0.19. This segment is involved in the protruding long loop (residues 100 – 113) of 0.19. In the wheat inhibitors lacking activity against HSA (such as inhibitor 0.28, see below), this sequence is replaced by the sequence Pro– Asn – Pro. It was suggested that this loop region, occupying a cavity on the surface of HSA, was likely to play a pivotal role in conferring the specificity of these inhibitors in a-amylases of both mammalian and insect origin [42].
3.4.4. Interaction of the wheat inhibitor 0.28 with the insect a-amylase TMA ˚ by molecular The crystal structure was solved at 2.9 A replacement using the structure of free TMA (PDB ID-code, 1JAE) as the search model (F. Payan, V. Nahoum, S. Strobl, R. Glockshuber, R. Huber, E. Poerio, unpublished data). The overall 3-D structure of the wheat monomeric inhibitor 0.28 is similar to that of 0.19 and RBI. Four helices (a1 to a4) with an ‘‘up and down’’ topology constitute the main body of the molecule. The helices are linked together by loop segments, L1 (residues 30– 34), L2 (residues 47 –55), and L3 (residues 68 –82). The C-terminal loop L4 (residues 99– 113) follows after helix a4. All 10 cysteine residues in 0.28 form disulfide bonds, occurring in the pairs: Cys7 – Cys54, Cys21 – Cys42, Cys29 – Cys82, Cys43 – Cys – 98, and Cys56 – Cys113 (as previously reported [48]). The pattern of disulfide bridges in 0.28 is structurally identical to that occurring in 0.19 [44]: Cys6– Cys52, Cys20 –Cys41, Cys28– Cys83, Cys42– Cys99, and Cys54– Cys115. The N-terminal segment, in a disordered state in the Xray structure of free 0.19, is clearly defined in the structure of 0.28 in complex with TMA: in the helical conformation, it curls into the active site V-shaped depression. Likewise, the five N-terminal residues, flexible in the solution structure of free RBI, adopted a 310-helical conformation in the complex RBI – TMA and filled the substrate binding site of the enzyme. In loops L3 and L4 of the 0.28 –TMA structure, two segments: residues 68– 82 (from L3) and 106 –110 (from L4) were found in a disrupted electron density pattern. In the free structure of the wheat inhibitor 0.19: a similar L3 conformation was observed, the loop containing many disordered residues, while L4 was clearly defined and described as a long loop region (residues 102 – 113) including short antiparallel h-strands protruding into the solvent. Loop L3, in 0.28, may be able to adopt multiple conformations, as proposed in the case of the corresponding loop of inhibitor 0.19 [44]. In the C-terminal region of the 0.28 inhibitor (in complex with TMA), residues 120– 123 were not present in the electron density map. The same structural
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feature was also observed with the last five residues of RBI in its complex with TMA [6] and, as mentioned above, in the modeled interaction of 0.19 with TMA [42], the residues located at the C-terminal end of the inhibitor molecule, superimposed with the facing enzyme region. In the RBI – TMA structural analysis [6], the authors proposed that access of substrate may also be prevented by steric hindrance conferred by these C-terminal residues. Thus, it is tempting to suggest that this may be a general mechanism for these a-amylase inhibitors of the cereal a-amylase superfamily. The 3-D structure of the complex 0.28 –TMA shows three regions of contact: the N-terminal segment (residues 1 –10); residue 53 (from L2), and the region including residues 103– 119 (from L4). The N-terminal segment of 0.28 fills the central substrate-binding subsites of TMA and targets the catalytic residues of the enzyme. Although some differences were found in the amino acid sequence of this region, a similar network of interactions to that observed in the substrate binding cleft of the RBI – TMA structure (segments 1 – 5 in Ref. [6]) is conserved in the 0.28 – TMA structure (segments 1 – 6). The main structural differences between the wheat inhibitors 0.19 and 0.28 occur in the N-terminal and C-terminal regions. The structure-segment 4 – 15 is differently shaped (residues 1– 4 are not present in the structure of free 0.19). The protruding hairpin loop (L4) of 0.19 (sequence Val – Val – Asp – Ala from residues 104 – 107; see Section 3.4.2) is replaced in 0.28 by a shorter segment in which the corresponding sequence is Pro –Asn – Pro (see Fig. 4). L4 established contact with the domain B of the enzyme and with non-reducing end substrate-binding subsites. Overall, the 3-D structure of the wheat inhibitor 0.28 is similar to that of 0.19 and RBI [6,44]. However, despite very similar individual structures, superimposition of the X-ray structures of the complexes RBI – TMA and 0.28– TMA (via superimposition of the TMA part) showed that the orientations of RBI and 0.28 in the complexes were significantly different (Fig. 4). These results weaken the predicted importance of the two first areas of interest deduced from the 0.19 – HSA modeling (see paragraph Section 3.4.2) because of their obvious dependence on the orientation of the inhibitor. On the contrary, the substantial differences, present in the C-terminal region of the two wheat inhibitor structures, strengthen the suggestion from Franco et al. [42] that the loop region L4, plays an important role in the specificities of the 0.28 and 0.19 wheat inhibitors. In summary, the reported structures of these complexes have provided insights into some general aspects of the inhibition strategy of mammalian and insect a-amylases: (i) the inhibition occurs mainly via interactions within the enzyme substrate binding site, the aromatic residues lining the active site play an important role; (ii) the subsites are usually occupied by structural elements (side-chains and/or structure-segments) originating from the inhibitor molecule;
(iii) the structure-segments and loop regions strongly involved in the inhibition process are likely to correspond to flexible components of the free structures of the molecules; (iv) ‘‘dual’’ inhibitors specific to both mammalian and insect a-amylases act through a common mechanism using alternative ways. Comparisons between the interaction patterns involved in the structural modes of a-amylase inhibition by inhibitors and analyzing the subtle differences which occur should aid all those engaged in designing and synthesizing a-amylase inhibitors to serve as therapeutic agents or tools for crop plant protection. The wide variety of a-amylase complex structures provides a whole ‘‘textbook’’ covering the most typical aspects of the inhibition mechanisms which can serve to predict those which are likely to occur in other a-glycosidase complexes.
Acknowledgements I would like to thank all those who contributed to this study and agreed to mention unpublished data in this minireview. My thanks go to Dr. Nathalie Juge (IFR Norwich, UK) for critical reading of the manuscript before submission. I would also like to thank Dr. A. Roussel (AFMB Marseille) for helpful assistance.
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Review
Structural basis for the inhibition of mammalian and insect a-amylases by plant protein inhibitors Francßoise Payan * Architecture et Fonction des Macromole´cules Biologiques, UMR6098, CNRS and Universities Aix-Marseille I and II, 31 Chemin Joseph Aiguier, F-13402 Marseilles, France Received 8 May 2003; accepted 23 October 2003
Abstract a-Amylases are ubiquitous proteins which play an important role in the carbohydrate metabolism of microorganisms, animals and plants. Living organisms use protein inhibitors as a major tool to regulate the glycolytic activity of a-amylases. Most of the inhibitors for which three-dimensional (3-D) structures are available are directed against mammalian and insect a-amylases, interacting with the active sites in a substrate-like manner. In this review, we discuss the detailed inhibitory mechanism of these enzymes in light of the recent determination of the 3-D structures of pig pancreatic, human pancreatic, and yellow mealworm a-amylases in complex with plant protein inhibitors. In most cases, the mechanism of inhibition occurs through the direct blockage of the active center at several subsites of the enzyme. Inhibitors exhibiting ‘‘dual’’ activity against mammalian and insect a-amylases establish contacts of the same type in alternative ways. D 2003 Elsevier B.V. All rights reserved. Keywords: a-Amylase; Inhibitor; Bean inhibitor; Cereal inhibitor; Wheat inhibitor; X-ray structure
1. Introduction a-Amylases (a-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) are a group of enzymes widely distributed in microorganisms, plants and animal secretions which catalyze the hydrolysis of the (a-1,4) glycosidic linkages in starch and various oligosaccharides. Carbohydrate normally constitutes the main component of the human diet and starch accounts for most of the ‘carbohydrate pool’. The cleavage of starch by a-amylases constitutes the first step in the enzymatic degradation of polysaccharides which is essential
Abbreviations: 0.28 and 0.19, a-amylase proteinaceous inhibitors from wheat kernel with molecular weights of 12,000 and 24,000 and gelelectrophoretic mobilities, relative to bromophenol blue, of 0.28 and 0.19, respectively; TMA, a-amylase from the yellow mealworm (larvae of Tenebrio molitor); HPA, human pancreatic a-amylase; HSA, human salivary a-amylase; PPA, pig pancreatic a-amylase; a-AI, a-amylase inhibitor from bean Phaseolus vulgaris; RBI, bifunctional a-amylase/ trypsin inhibitor from ragi (Eleusine coracana Gaertneri, Indian finger millet); AAI, a-amylase inhibitor from Amaranthus hypocondriacus seeds; r.m.s.d., root mean square deviation; PDB ID-code, Brookhaven Protein Data Bank accession reference * Tel.: +33-4-9116-4506; fax: +33-4-9116-4536. E-mail address: [email protected] (F. Payan). 1570-9639/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2003.10.012
in carbohydrate assimilation. Inhibition of a-amylases reduces the post-prandial glucose peaks, which is of particular importance in patients with diabetes. Carbohydrate in the form of starch also forms the bulk of the diet of insects such as the meal beetle Tenebrio molitor and its larvae, the yellow mealworm. These insects are cosmopolitan pests feeding on seed products. Plants have evolved defence strategies to counteract these effects through enzyme inhibitors impeding the action of insect gut digestive a-amylases and proteinases. Understanding the inhibitory mechanisms exerted on mammalian and insect a-amylases is a key step in the design of high affinity/selectivity a-amylase inhibitors having potentials in various fields, from the treatment of diabetes to crop protection. The number of proteinaceous a-amylases inhibitors isolated and identified so far is extremely large. They occur in microorganisms, higher plants and animals [1]. Proteinaceous a-amylases inhibitors can have different polypeptides scaffolds and can be grouped by their tertiary structures into six classes: lectin-like, knottin-like, cerealtype, Kunitz-like, g-purothionin-like and thaumatin-like. Plant a-amylase inhibitors, which are abundant in cereals and leguminosae, have been extensively studied. The area has benefited from the recent determination of many struc-
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tures of a-amylases, inhibitors and complexes: see Ref. [2] for a review describing the properties of insect a-amylases and their inhibitors. Three-dimensional (3-D) structures of plant protein inhibitors in complex with a-amylases involve a lectin-like a-amylase inhibitor from bean (Phaseolus vulgaris): (a-AI) [3– 5], a cereal-type a-amylase inhibitor: bifunctional a-amylase/trypsin inhibitor from ragi (Eleucine coracana Gaertneri; Indian Finger Millet) (RBI) [6], a knottin-type a-amylase inhibitor, amaranth a-amylase inhibitor (AAI) [7]; a Kunitz-like a-amylase inhibitor, barley a-amylase/subtilisin inhibitor (BASI) [8]. This review is aimed at displaying structural characteristics of the inhibition mechanism of mammalian and insect a-amylases by plant inhibitors by way of examples Refs. [3 –7] and also reports 3-D structure of an insect a-amylase in complex with the monomeric wheat inhibitor 0.28 (cereal-type). The mechanism of inhibition of the endogenous barley a-amylase 2 (AMY2) by BASI [8] is reported elsewhere in this issue.
2. Interaction of carbohydrate inhibitors with mammalian and insect A-amylases: structural basis for their catalytic mechanism Insights into the catalytic mechanism of mammalian aamylases came from the resolution of 3-D structures of PPA and human pancreatic a-amylase (HPA) in complex with carbohydrate inhibitors. Agents having inhibitory effects on PPA and on the sucrase and maltase activities of intestinal disaccharidases were first discovered in vitro in actinomycetes broths [9]. These substances correspond to homologous pseudo-oligosaccharide compounds (trestatin family), which are by now well-known. They contain a specific structural entity named acarviosine (an unsaturated cyclitol unit linked to a 4,6-dideoxy-4-amino-D-glucose) which is the essential structural unit responsible for the inhibitory function of this family of inhibitors. This unit is linked by a-(1,4)-O-glycosidic bonds to a variable number of glucose residues. One particular compound, the pseudotetrasaccharide acarbose, in which the acarviosine unit is linked to a maltose molecule [9], is a potent inhibitor of aamylases used to treat diabetic diseases. The architecture of mammalian and insect a-amylases consists of three domains: the catalytic core domain (A), comprising a (h/a)8 barrel, contains an extended loop inserted between the third h-strand and the third a-helix (called domain B). The C-terminal domain (domain C) forms a distinct globular unit forming a Greek key motif (Fig. 1). Both mammalian and insect enzymes require one essential calcium ion to maintain their structural integrity, and are activated by chloride ions. Elements from domain A and B are involved in the architecture of the three most functionally important sites: the active site, the calcium-binding site, and the chloride-binding site [10 –13].
Fig. 1. MOLSCRIPT [49] diagram of the a-amylase (PPA) structure. Domain A is shown in red, domain B in yellow and domain C in purple. The calcium ion (blue sphere) and the chloride ion (yellow sphere) are also shown in the immediate vicinity of the catalytic center. An acarbose-derived ligand (ball-and-stick representation in green) is bound at the active site cleft. Monosaccharide and disaccharide ligands (in ball-and-stick representation) are shown bound to the surface binding sites [50].
An atomic description of the interactions occurring within the active site depression between PPA and carbohydrate inhibitors have been published along with the refined structures of PPA – acarbose complexes [14,15] (determined using two different crystal forms), that of PPA complexed with V-1532, a member of the trestatin family [16] and that of HPA interacting with trestatin-derived compounds (acarbose and B4) [5,17]). The strong inhibition of a-amylases by these compounds is mainly attributed to the binding at subsite 1 (where the reaction intermediate is formed) of the cyclitol unit. In half-chair conformation it mimics the substrate distortion expected to occur in the transition state (for a review on possible reaction mechanisms for glycosidases, see Ref. [18]). The adjacent glycosidic bond is N-linked and prevents enzymatic hydrolysis; acid hydrolysis of acarbose leads to the breakage of both O-glycosidic bonds while enzymatic hydrolysis by a-amylase only results in the cleavage of the linkage between the two glucose units [19].
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More recently, the high-resolution structure of PPA complexed with the acarviosine-glucose member of the trestatin family was solved [20]. It was shown that an hexasaccharide species, filling subsites 4 though + 2 and composed of two a-1,4-linked original molecules remained bound to the enzyme active site (Fig. 2; PDB ID-code, 1HX0). These Xray structures clearly show a subset of residues in PPA active site directly involved in binding the inhibitor. The structural arrangement observed upon binding of the carbohydrate inhibitors indicates that PPA possesses all the requirements for hydrolysis to occur via the general acid hydrolysis mechanism. The widely recognized two-step mechanism originally proposed by Koshland [21] for retaining glycoside hydrolases requires the presence of two carboxyl-containing amino acids, the acid/base catalyst and the nucleophile, responsible for the formation of the glycosyl – enzyme intermediate [22]. The crystallographic data of PPA in complex with acarbose suggested that both Asp197 and Glu233 are required to produce the h-linked glycosyl – enzyme intermediate [14]. McCarter and Withers [23] later confirmed that Asp197 serves as the catalytic nucleophile. In addition, Xray crystallographic studies on a family 13 cyclodextrin glucanotransferase (CGTase) mutant (Glu257Gln), trapped as a glycosyl – enzyme intermediate via a reaction with 4deoxymaltotriosyl a-fluoride, showed the existence of an unequivocal h-glycosidic covalent bond located between the Asp229 Oy1 atom (corresponding to Asp197 in PPA) and the substrate anomeric C1 atom [24]. In the trestatin-derived
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PPA and HPA complexes, Glu233 and Asp300 are hydrogen-bonded to each other via an intervening water molecule, as well as to the nitrogen atom of the non-hydrolyzable Nglycosidic bond. Glu233, located in close proximity to the chloride ion and an intervening water molecule, is the most appropriate candidate for the role of general acid/base catalyst as previously substantiated by the results of structural analysis [14,25] and recently confirmed by Rydberg et al. [26]. Insect and mammalian a-amylases display high homology in their primary and tertiary structures. For example, a-amylase from the yellow mealworm (TMA) and porcine pancreatic a-amylase (PPA) share 54% sequence identity [27], and 410 structurally conserved Ca atoms super˚ [13]. Based on PPA imposed with an r.m.s.d. of 2.2 A crystallographic data [16], a model for the binding of a pseudo-hexasaccharide ligand in the active site depression of TMA has been constructed [6]. This model indicates that TMA can accommodate six saccharide units. Asp185, Glu222, and Asp287 (corresponding to Asp197, Glu233, and Asp300 in PPA) are proposed as key residues for catalysis [13]. It is likely that both mammalian and insect a-amylases have a similar mechanism of action. More recently, the X-ray structures of plant protein inhibitors in complex with either the pancreatic a-amylases HPA and PPA, or TMA have shed light on the structural basis for the inhibitory mechanisms of mammalian and insect a-amylases.
Fig. 2. Electron density of an acarbose-derived ligand bound to the 4 to + 2 subsites of PPA [20]. In the acarbose-derived extended ligand, the cyclitol unit is bound at subsite 1 (where the reaction intermediate state is formed); nomenclature n/ + n, assuming the cleavage to take place between subsites 1 and ˚ (at 1.38 A ˚ resolution). The figure was prepared using + 1, is according to Ref. [51]. The map shown is a 2Fo Fc electron density contoured at 0.5 10 3 A TURBO-FRODO [52].
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3. Interaction of plant proteinaceous inhibitors with mammalian and insect A-amylases—structural basis for the inhibition mechanism 3.1. The bean P. vulgaris a-amylase inhibitor The seeds of the common bean, P. vulgaris, contain a family of plant defense proteins including an a-amylase inhibitor, a-AI [28]. Two forms of the a-amylase inhibitor have been described, a-AI1 and a-AI2 which differ in both their primary sequence and their inhibitory activity towards bruchids [29 –31]. a-AI1 is a plant defence protein and a potent inhibitor of both mammalian and insect a-amylases (for PPA, Kd = 3.5 10 11 M at neutral pH) [32]. The overall architecture of the bean-inhibitor corresponds to a classical lectin fold [3] (PDB ID-code, 1DHK). The X-ray structures of the inhibitor in complex with either HPA, PPA, or TMA have been determined [3 –5]. 3.1.1. Interaction of a-AI1 with the mammalian a-amylases HPA and PPA The main interactions with PPA and HPA occur directly at the V-shaped depression of the a-amylase active site, which is also the binding site for acarbose [14]. The inhibition process is very similar for both enzymes. In both a-AI1 –HPA and aAI1 – PPA complexes, two facing hairpin loops emerging from the h-sheet fold of the inhibitor lie fully in the active site depression forming extensive hydrogen-bonding, hydrophobic and water-bridged contacts with the active site residues of the enzyme. At the heart of the substrate-binding site, two tyrosine residues (Tyr37 and Tyr186) protruding from the extremity of the two hairpin loops of a-AI combine to provide interactions with the catalytic residues, nucleophile and acid catalyst of the enzyme (Asp197 and Glu233, respectively). The interactions occurring in the region of subsites 1, + 1, + 2 are highly conserved in the complexes between carbohydrate or proteinaceous inhibitors and pancreatic a-amylases (PPA and HPA). Hydrophobic interactions also occur between the substrate’s surface and the hydrophobic residues lining the entrance of the cleft (subsite 2, 3). These interactions are followed by protein – protein interactions involving areas further away from the catalytic center, namely the loop 303 – 312, the loop at position 237 –240, the loop 347 – 357, and the loop 140 – 150 from domain B. Although the mechanisms of inhibition of HPA and PPA by a-AI are very similar, a close inspection of the structure of the a-AI1– HPA and a-AI1– PPA complexes showed the presence of additional hydrogen bonds between the inhibitor and domain B of the human enzyme. PPA and HPA show discrepancy in their amino acid sequences in this area which are also believed to influence the substrate specificity of the two enzymes [5]. A different network of interactions was also observed in the loop regions 303 – 312 and 347– 357 of domain A.
The structural changes induced in response to the binding of a-AI at the active site of the enzyme are very different from those induced by the carbohydrate inhibitor acarbose. The main displacement occurring upon the binding of the carbohydrate inhibitors involves the ‘‘flexible loop’’ (residues 303 – 309) [14]. An additional area that undergoes significant conformational changes is the loop extending from residues 237 –240, which forms the surface edge at the substrate-reducing end periphery of the active site depression. Residues from this loop and from the ‘‘flexible loop’’ are involved in the architecture of subsite + 2. Also affected by the carbohydrate inhibitor binding process is the loop segment at position 140 –150 of the domain B [5,15,20]. Two particular acarbose-binding residues, the catalytic residue Asp300 and residue His305 undergo ˚ takes place substantial changes. A movement of about 5 A at residue His305 which approaches the acarbose-derived ligand from the solvent side and forms a strong hydrogen bond with the residue in subsite 2. The side-chain of the catalytic residue Asp300 undergoes an induced conformational change upon substrate binding. It rotates about 60j around the Ca –Ch bond and becomes hydrogen bonded to the acid catalyst Glu233 via an intervening water molecule. Upon substrate binding, the ‘‘flexible loop’’ moves in toward the saccharide, thus reducing the cleft breadth. In contrast, when complexed with a-AI, the same loop moves out toward the solvent, pushed away by the inhibitor as a result of the tight-binding inhibition process. This movement is accompanied by the readjustment of the surrounding regions; in particular, the loop region including residues 351 – 359 in domain A. The side chain of the catalytic residue Asp300 shows the same orientation in both the free structure of PPA and the a-AI – PPA complex structure (instead of its substrate-induced conformation), suggesting that Asp300 adopts this functional position when a sugar unit is bound at subsite 1. In summary, the inhibitor a-AI completely blocks the substrate-reducing end of the enzyme cavity and prevents access to the other end via a steric hindrance process. The inhibitor triggers substrate ‘‘mimetic’’ interactions with the binding subsites on the enzyme and all the catalytically competent components of the enzyme are targeted (as an example, see Fig. 3a and b). 3.1.2. Interaction of a-AI1 with the insect a-amylase TMA Although TMA has the structure typical of a-amylases [13] (PDB ID-code, 1JAE) with an active site showing a V-shaped depression very similar to that of pancreatic aamylases, large deviations occur in the loops and particularly in the loop segments next to the active site region. However, structural analyses of the complex between aAI1 and TMA [4] (PDB ID-code, 1viw) indicated that the strong contacts occurring between the inhibitor and the catalytic cleft of the PPA, HPA and TMA enzymes are highly conserved. The two hairpin loops of the inhibitor
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residues Tyr186 and Tyr37, which project into the heart of the catalytic site and to the flanking residue Asp38, which occupies the subsite + 2 and blocks the access to the region of the ligand-reducing end. In summary, despite differences occurring in the interacting loops of the enzymes, a-AI1 inhibits both the insect and mammalian a-amylases via the same inhibitory mechanism. The protein partners being able to produce the same type of contacts in alternative ways. 3.2. The ragi bifunctional inhibitor The bifunctional a-amylase/trypsin inhibitor RBI from ragi (E. coracana Gaertneri; Indian finger millet) is the prototype of a cereal inhibitor superfamily. It is a monomer of 122 amino acids [33] with five disulfide bonds. It is a potent inhibitor of TMA (Ki = 15 nM) [6,34] and PPA (Ki = 11 nM for PPAII isoform; [34]). The 3-D structure of RBI was solved using nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography procedures [35,36]. The globular fold of RBI consists of four helices with a simple ‘‘up and down’’ topology and a small antiparallel h-sheet (PDB ID-code, 1B1U).
Fig. 3. (a) A trestatin-derived inhibitor bound to the active site of HPA. The five occupied subsites are labeled 3 to + 2. The catalytic residues, nucleophile and acid/base catalyst are all shown in red; model produced using GRASP [53]. (b) Selected residues of the proteinaceous inhibitor aAI interacting in the active site of HPA. The figure was adapted from Ref. [5]. Similar views of the TMA active site comparing interaction with: (a) a pseudo-hexasaccharide inhibitor V-1532 [16], and (b) RBI and AAI residues are available in Refs. [6,7], respectively.
(residues 29 –46 and 171 –189), which protrude into the active site of TMA, establish a similar network of hydrogen bonds, with the residues of the substrate-docking region, to that observed in the complexes between pancreatic a-amylases and a-AI1. The catalytic residues of TMA are strongly hydrogen-bonded to the inhibitor
3.2.1. Interaction of RBI with the insect a-amylase TMA In the RBI – TMA complex (PDB ID-code, 1TMQ) [6], the inhibitor binds to the active site of the enzyme. As observed with the bean inhibitor a-AI1, RBI interacts with residues from domains A and B which line the substrate binding site of TMA. In the RBI –TMA complex, the subsites of TMA (defined by analogy with those of PPA) are completely blocked by RBI residues. Inhibition by RBI mainly requires the harrow-head-like segment composed of N-terminal residues Ser1 – Ala11 and residues Pro-52 – Cys-55, which protrude into the TMA substrate binding groove and directly target the catalytic residues [6]. It is worth noting that although residues 1 –5 are flexible in the solution structure of free RBI, they adopt a 310-helical conformation in the complex, filling the substrate binding site of TMA. As explained above, the binding of a-AI to PPA and to TMA occurs through very similar contacts to residues in and around the active site cleft. The interaction between the shortened loops (corresponding to ‘deleted’ regions) of TMA and a-AI being maintained via neighboring TMA residues. In view of these results, the inhibition strategies for two other proteinaceous inhibitors, RBI and Tendamistat (a proteinaceous inhibitor from Streptomyces) against PPA and TMA have been compared [6,7]. Models of RBI – PPA and of Tendamistat– TMA complexes were constructed on the basis of the X-ray structures of the complexes RBI – TMA [6] and Tendamistat – PPA [37], respectively. Structural comparisons of these models with the respective experimentally determined structures led to the conclusion, similar to that of Nahoum et al. [4], that all three proteinaceous inhibitors
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(RBI, a-AI and Tendamistat) can inhibit both mammalian and insect a-amylases using alternative ways [7]. 3.3. The amaranth a-amylase inhibitor AAI specifically inhibits a-amylases from insects, but not from mammalian sources [38]. With 32 amino acids, AAI is the smallest a-amylase inhibitor described so far. Based on its overall fold, its three-stranded twisted hsheet and the topology of its disulfide bonds, AAI has been classified as a knottin-like protein (for a detailed bibliography, see Refs [2,7]). 3.3.1. Interaction of AAI with the insect a-amylase TMA The crystal structure of TMA in complex with AAI ˚ resolution (PDB ID-code, 1CLV) was determined at 2.0 A [7]. The inhibitor binds into the active site depression of TMA, blocking the central four sugar-binding subsites. The structure of the AAI – TMA complex shows that inhibition, as for the lectin-like inhibitor (a-AI), occurs through blockage of the substrate-binding sites by residues from AAI targeting the active site residues of TMA [7]. In the interaction of AAI with TMA, only Asp287, one of the catalytic residues of TMA (corresponding to Asp300 in PPA) forms a salt bridge directly with the inhibitor residue Arg7. The other catalytic residues: the nucleophile and acid catalyst, Asp185 and Glu222, respectively (by analogy with PPA residues Asp197 and Glu233), are connected via an intricate water-mediated hydrogen-bonding network to the inhibitor residues. In contrast, in the inhibition scheme of TMA by a-AI and RBI, all three catalytic residues establish direct hydrogen bonds with the functional groups of inhibitor residues. The structural analysis of the AAI – TMA complex [7] provides insights into the lack of recognition of mammalian a-amylases [38]. In particular, the extended network of hydrophobic interactions observed in other complexes of a-amylases and proteinaceous inhibitors [3,6,37] could not be identified [7]. Comparison of the modeled AAI – PPA complex with the AAI – TMA complex X-ray structure identified six hydrogen bonds specific for the AAI – TMA complex and probably responsible for the high specificity of the AAI inhibitor against insect a-amylase. 3.4. The wheat inhibitors Wheat kernels are particularly richly endowed with aamylase inhibitors affecting both insect and mammalian enzymes. Some of these substances specifically inhibit insect enzymes, in that they inhibit a-amylases from insects strongly but inhibit mammalian a-amylases only weakly or not at all. A set of a-amylase inhibitors were purified from wheat flour: they display molecular masses of about 12, 24, and 60 kDa and are members of the cereal superfamily of a-amylase inhibitors. They act as monomers, homodimers or hetero-oliogomers.
The insect-specific a-amylase inhibitors are mostly monomeric proteins (members of the 12 kDa family); the members from the 24 kDa family actively inhibit both mammalian and insect a-amylases [39]. The T. molitor aamylase (TMA) is effectively inhibited by a number of water-soluble protein components of the wheat kernel, particularly those termed inhibitors 0.28 and 0.19, which refers to their gel electrophoretic mobility relative to bromophenol blue [39,40]. The inhibitor 0.28 is a monomer with a molecular weight of 12 kDa, whereas the inhibitor 0.19 is a dimer with a molecular weight of about 24 kDa [40]. The dissociation constants of the a-amylase inhibitor 0.19 and a-amylase inhibitor 0.28 complexes are 0.85 and 0.13 nM, respectively [41]. 3.4.1. The wheat inhibitor 0.19 The inhibitor referred to as 0.19 inhibits a-amylases from human saliva, pig pancreas, chick pancreas, the yellow mealworm, and Bacillus subtilis [42]. The molecule has 124 amino acid residues [43] and its amino acid sequence is 26% identical to that of RBI [33]. It is the only other member of the cereal family with a known 3-D structure (PDB ID-code, 1HSS) [44]. The crystallography study revealed that 0.19 is an homodimer in accordance with the previous results obtained by gel chromatography [40,45]. In the crystal structure, the asymmetric unit has four molecules of 0.19, each corresponding to a monomer of 124 amino acid residues. Each subunit consists of four major a-helices arranged in an ‘‘up and down’’ pattern and two short antiparallel h-strands [44]. The h-strands are included in a characteristic protruding hairpin loop inserted before a one-turn helix (helix-5) into the Cterminal region of the molecule. The electron density for two regions including residues 1– 4 and 68 – 78 was not present in all four subunits, suggesting that these residues have multiple conformational states [44]. The 3D structure of the 0.19 subunits appears to be very similar to that of RBI, although two characteristic loops differ markedly. Firstly, the protruding segment which occurs in RBI between helices a3 and a4 (including short antiparallel h-strands) is absent from the structure of 0.19 (Fig. 4) for which, most of the residues in this loop region are disordered (residues 68 –78). Secondly, the characteristic protruding loop present in 0.19 on the other side of the molecule (where the C-terminal segment is located) is more extensive than the corresponding loop in RBI. 3.4.2. Interaction of the wheat inhibitor 0.19 with mammalian and insect a-amylases In order to identify the factors determining the specificity of the wheat inhibitors acting on mammalian and insect a-amylases, the overall structural similarity between RBI and 0.19 was used to model the interaction of 0.19 with TMA and HSA [42]. Despite some steric clashes between a few side-chains from the two partners, 0.19 inhibitor could be readily accommodated in the binding
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3.4.3. The wheat inhibitor 0.28 The monomeric wheat a-amylase inhibitor known as 0.28 is one of the most fully characterized inhibitor of this kind. It is highly active against a-amylase of T. molitor (TMA) and only weakly inhibits the a-amylases from human saliva and pancreas and from the pancreas of some avians [39]. The 123 amino acids sequence [46,47] is 60% identical to that of 0.19 and 26% to that of RBI. All 10 cysteine residues in the molecule form disulfide bridges [48]. The 3-D structure of the complex between TMA and 0.28 has recently been solved (F. Payan, V. Nahoum, S. Strobl, R. Glockshuber, R. Huber, E. Poerio, unpublished results). Below are some of the structural characteristics of this complex.
Fig. 4. Interaction of the cereal inhibitors with the substrate binding cleft of TMA. (A, top) The molecular surface of TMA is shown in grey. The main chains of RBI, 0.28 and 0.19 are represented by grey, green, and red tubes, respectively. The free structure of 0.19 is superimposed onto the structure of 0.28 (as bound to TMA). The loop L4 is circled; the N-terminal segment is only present in the X-ray structures of RBI and 0.28 in complex with TMA (see text). (B, bottom) A close-up of the loops L4 as observed in 0.19 (in red) and 0.28 (in green), as bound to TMA, is shown. Both figures were made with SPOCK [54].
site of the enzymes in the same orientation as seen for RBI. At the C-terminus of 0.19, the two residues 123 – 124 superimposed with the facing enzyme region and were therefore deleted from the model. The inhibitor interface residues that differ between 0.19 and other wheat a-amylase inhibitors of this family (see Ref. [42] for a detailed nomenclature) were analyzed using the modeled complex of HSA-0.19. As a result, three inhibitor spots of interest were proposed. The first is residue His47, situated, in the model, near residue Glu349 of HSA; the second concerns Ser49 of 0.19, closely packed by Lys352, and Asp356 of the long loop of HSA; the third region of interest is the sequence Val – Val – Asp –Ala from residues 104 –107 of 0.19. This segment is involved in the protruding long loop (residues 100 – 113) of 0.19. In the wheat inhibitors lacking activity against HSA (such as inhibitor 0.28, see below), this sequence is replaced by the sequence Pro– Asn – Pro. It was suggested that this loop region, occupying a cavity on the surface of HSA, was likely to play a pivotal role in conferring the specificity of these inhibitors in a-amylases of both mammalian and insect origin [42].
3.4.4. Interaction of the wheat inhibitor 0.28 with the insect a-amylase TMA ˚ by molecular The crystal structure was solved at 2.9 A replacement using the structure of free TMA (PDB ID-code, 1JAE) as the search model (F. Payan, V. Nahoum, S. Strobl, R. Glockshuber, R. Huber, E. Poerio, unpublished data). The overall 3-D structure of the wheat monomeric inhibitor 0.28 is similar to that of 0.19 and RBI. Four helices (a1 to a4) with an ‘‘up and down’’ topology constitute the main body of the molecule. The helices are linked together by loop segments, L1 (residues 30– 34), L2 (residues 47 –55), and L3 (residues 68 –82). The C-terminal loop L4 (residues 99– 113) follows after helix a4. All 10 cysteine residues in 0.28 form disulfide bonds, occurring in the pairs: Cys7 – Cys54, Cys21 – Cys42, Cys29 – Cys82, Cys43 – Cys – 98, and Cys56 – Cys113 (as previously reported [48]). The pattern of disulfide bridges in 0.28 is structurally identical to that occurring in 0.19 [44]: Cys6– Cys52, Cys20 –Cys41, Cys28– Cys83, Cys42– Cys99, and Cys54– Cys115. The N-terminal segment, in a disordered state in the Xray structure of free 0.19, is clearly defined in the structure of 0.28 in complex with TMA: in the helical conformation, it curls into the active site V-shaped depression. Likewise, the five N-terminal residues, flexible in the solution structure of free RBI, adopted a 310-helical conformation in the complex RBI – TMA and filled the substrate binding site of the enzyme. In loops L3 and L4 of the 0.28 –TMA structure, two segments: residues 68– 82 (from L3) and 106 –110 (from L4) were found in a disrupted electron density pattern. In the free structure of the wheat inhibitor 0.19: a similar L3 conformation was observed, the loop containing many disordered residues, while L4 was clearly defined and described as a long loop region (residues 102 – 113) including short antiparallel h-strands protruding into the solvent. Loop L3, in 0.28, may be able to adopt multiple conformations, as proposed in the case of the corresponding loop of inhibitor 0.19 [44]. In the C-terminal region of the 0.28 inhibitor (in complex with TMA), residues 120– 123 were not present in the electron density map. The same structural
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feature was also observed with the last five residues of RBI in its complex with TMA [6] and, as mentioned above, in the modeled interaction of 0.19 with TMA [42], the residues located at the C-terminal end of the inhibitor molecule, superimposed with the facing enzyme region. In the RBI – TMA structural analysis [6], the authors proposed that access of substrate may also be prevented by steric hindrance conferred by these C-terminal residues. Thus, it is tempting to suggest that this may be a general mechanism for these a-amylase inhibitors of the cereal a-amylase superfamily. The 3-D structure of the complex 0.28 –TMA shows three regions of contact: the N-terminal segment (residues 1 –10); residue 53 (from L2), and the region including residues 103– 119 (from L4). The N-terminal segment of 0.28 fills the central substrate-binding subsites of TMA and targets the catalytic residues of the enzyme. Although some differences were found in the amino acid sequence of this region, a similar network of interactions to that observed in the substrate binding cleft of the RBI – TMA structure (segments 1 – 5 in Ref. [6]) is conserved in the 0.28 – TMA structure (segments 1 – 6). The main structural differences between the wheat inhibitors 0.19 and 0.28 occur in the N-terminal and C-terminal regions. The structure-segment 4 – 15 is differently shaped (residues 1– 4 are not present in the structure of free 0.19). The protruding hairpin loop (L4) of 0.19 (sequence Val – Val – Asp – Ala from residues 104 – 107; see Section 3.4.2) is replaced in 0.28 by a shorter segment in which the corresponding sequence is Pro –Asn – Pro (see Fig. 4). L4 established contact with the domain B of the enzyme and with non-reducing end substrate-binding subsites. Overall, the 3-D structure of the wheat inhibitor 0.28 is similar to that of 0.19 and RBI [6,44]. However, despite very similar individual structures, superimposition of the X-ray structures of the complexes RBI – TMA and 0.28– TMA (via superimposition of the TMA part) showed that the orientations of RBI and 0.28 in the complexes were significantly different (Fig. 4). These results weaken the predicted importance of the two first areas of interest deduced from the 0.19 – HSA modeling (see paragraph Section 3.4.2) because of their obvious dependence on the orientation of the inhibitor. On the contrary, the substantial differences, present in the C-terminal region of the two wheat inhibitor structures, strengthen the suggestion from Franco et al. [42] that the loop region L4, plays an important role in the specificities of the 0.28 and 0.19 wheat inhibitors. In summary, the reported structures of these complexes have provided insights into some general aspects of the inhibition strategy of mammalian and insect a-amylases: (i) the inhibition occurs mainly via interactions within the enzyme substrate binding site, the aromatic residues lining the active site play an important role; (ii) the subsites are usually occupied by structural elements (side-chains and/or structure-segments) originating from the inhibitor molecule;
(iii) the structure-segments and loop regions strongly involved in the inhibition process are likely to correspond to flexible components of the free structures of the molecules; (iv) ‘‘dual’’ inhibitors specific to both mammalian and insect a-amylases act through a common mechanism using alternative ways. Comparisons between the interaction patterns involved in the structural modes of a-amylase inhibition by inhibitors and analyzing the subtle differences which occur should aid all those engaged in designing and synthesizing a-amylase inhibitors to serve as therapeutic agents or tools for crop plant protection. The wide variety of a-amylase complex structures provides a whole ‘‘textbook’’ covering the most typical aspects of the inhibition mechanisms which can serve to predict those which are likely to occur in other a-glycosidase complexes.
Acknowledgements I would like to thank all those who contributed to this study and agreed to mention unpublished data in this minireview. My thanks go to Dr. Nathalie Juge (IFR Norwich, UK) for critical reading of the manuscript before submission. I would also like to thank Dr. A. Roussel (AFMB Marseille) for helpful assistance.
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