75 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES OF ACETYLCHOLINESTERASE WITH THE NATURAL CHOLINESTERASE INHIBITORS FASCICULIN AND HUPERZINE A Israel Silman, Michal Harel, Mia Raves, and Joel L. Sussman Departments of Neurobiology and Structural Biology Weizmann Institute of Science Rehovoth 76100, Israel INTRODUCTION Acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by rapid hydrolysis of acetylcholine (ACh) (Quinn, 1987). Anticholinesterase agents are used in the treatment of various disorders (Taylor, 1990), and have been proposed as therapeutic agents for the management of Alzheimer's disease (Giacobini & Becker, 1991, 1994). Two such anti- cholinesterase agents, both of which act as reversible inhibitors of AChE, have been licensed by the FDA: tacrine (Gauthier & Gauthier, 1991), under the trade name Cognex, and, more recently, E2020 (Sugimoto et al., 1992), under the trade name Aricept. Several other anticholinesterase agents are at advanced stages of clinical evaluation. The active site of AChE contains a catalytic subsite, and a so-called 'anionic' subsite, which binds the quaternary group of ACh (Quinn, 1987). A second, 'peripheral' anionic site is so named since it is distant from the active site (Taylor & Lappi, 1975). Bisquaternary inhibitors of AChE derive their enhanced potency, relative to homologous monoquaternary ligands (Main, 1976), from their ability to span these two 'anionic' sites, which are ca. 14 A apart. The 3D structure of Torpedo AChE (Sussman et al., 1991) reveals that, like other serine hydrolases, it contains a catalytic triad. Unexpectedly, however, for such a rapid enzyme, the active site is located at the bottom of a deep and narrow cavity; this cavity was named the 'aromatic gorge', since >50% of its lining is composed of the rings of 14 conserved amino acids (Sussman et al., 1991; Axelsen et al., 1994). X -ray crystallographic studies of complexes of AChE with drugs of pharmacological interest can reveal which amino acid residues are important for binding the drug, and where space might exist for modifying the drug itself, information crucial for structure- Progress in Alzheimer's and Parkinson's Diseases edited by Fisher et al., Plenum Press, New York, 1998. 523
524 I. Silman et al. based drug design. Valuable information can also be achieved by site-directed mutagenesis (Harel et al., 1992). In earlier studies (Harel et al., 1993), we described the structures of complexes of Torpedo AChE (TcAChE) with three ligands of pharmacological interest: namely, edrophonium, a strong competitive AChE inhibitor (Wilson & Quan, 1958), whose pharmacological action is in the peripheral nervous system (Taylor, 1990); decamethonium, a bisquaternary ligand which is both a neuromuscular blocker and a cholinesterase inhibitor (Zaimis, 1976); and tacrine, already licensed as an anti-alzheimer drug (see above), which is also a strong reversible inhibitor (Heilbronn, 1961). Modelling had predicted that the principal interaction of the quaternary group of ACh would be with Trp84, via electrostatic interaction with the 1t electrons of its indole ring (Sussman et al., 1991), rather than with a cluster of acidic amino acids, as had been predicted previously (Nolte et al., 1980); such an assignment was also supported by affinity labelling (Weise et al., 1990). The crystallographic data fully confirmed this unexpected interaction (Harel et al., 1993). Furthermore, they revealed a prominent role for others of the conserved aromatic residues within the gorge. Thus the phenyl ring of Phe330 contributed substantially to the 'anionic' subsite of the active site, while the 'peripheral' anionic site, located at the top of the gorge, contained three aromatic residues, Tyr70, Tyr121 and Trp279. The interaction of the two quaternary groups of decamethonium, located at the top and the bottom of the gorge, was primarily with these two sets of aromatic residues (Harel et al., 1993). In the following, we describe the structure of two additional TcAChE-ligand complexes recently solved in our laboratory: with fasciculin-ii (FAS), a member of the threefinger polypeptide toxin family, which was isolated from mamba venom (Harel et al., 1995); and with (-)-huperzine A (HupA), an alkaloid purified from a moss used in Chinese herbal medicine (Raves et al., 1997). RESULTS AND DISCUSSION FAS-TcAChE Complex The venoms of elapid snakes, including the Asian cobras and kraits, as well as the African mambas, contain a number of small proteins, containing 60--70 amino acids, which displaya broad spectrum of toxic activities (Harvey, 1991). Among the best studied are the a-neurotoxins of the venoms of the kraits and cobras, such as a-bungarotoxin, from the Formosan krait, Bungarus multicinctus. which are potent and specific blockers of the nicotinic acetylcholine receptor (Changeux et al. 1970). Other toxins of this family have been shown to act as blockers of ion channels (Albrand et al., 1995), muscarinic agonists (Segalas et al., 1995) and anticholinesterases (Cervei\ansky et al., 1991). Despite their diverse biological activities, they display substantial sequence and structural homology. X-ray and NMR studies show that the toxins share a common structural motif: a core, containing four disulfide bridges, from which three loops protrude, roughly like the fmgers of a hand (Ie Du et al., 1992). Accordingly, they are known as the three-fingered toxin family (Wonnacott & Dajas, 1994). Superposition of their structures reveals that, whereas the structure of the central core is conserved, the orientation of the fingers can vary considerably (Albrand et al., 1995), suggesting that they serve as determinants of biological specificity. No three-dimensional structure of a complex of a three-fingered toxin with its target was, however, available. Whereas in previous cases, the AChE-ligand complex was obtained by soaking the ligand into crystals of the native enzyme, FAS is too large to permit such an approach. Accordingly, orthorhombic crystals of the complex were obtained from a solution containing
Crystallographic Studies on Complexes of Acetylcholinesterase 525 Figure t. Stoichiometric complex offasciculin-ii (FAS) with TcAChE. Shown is a ribbon diagram of the biological dimer, in which the two subunits interact via a 4-helix bundle and a disulfide bridge (not shown). The two FAS molecules, displayed as a line trace, are positioned over the top of the gorge leading to the active site of each subunit. stoichiometric (1:1) amounts of the purified TcAChE and offas purified from the venom of the green mamba (Dendroaspis angustieeps), and a data set was obtained which could be refined at 3.0 A resolution. The structure indeed reveals a stoichiometric complex, with one FAS molecule bound to each subunit of the TcAChE dimer (Fig. 1). FAS is bound on the surface of the subunit, at the 'peripheral' anionic site, thus sealing the top of the narrow gorge leading to the active site. A similar structure was reported independently, by Bourne et af. (1995), for a complex offas with mouse recombinant AChE. It has been noted previously that AChE has a large dipole moment (> 1 000 Debye), aligned approximately along the axis of the 'aromatic' gorge (Ripoll et al., 1993; Porschke et al., 1996). The field generated by this dipole might actually draw the positively charged substrate, ACh, down the gorge towards the active site. Similarly, FAS has its charges separated (dipole moment ea. 185 Debye), with most basic residues occurring in the first two fingers, which make intimate contact with TcAChE, and most acidic residues in the third finger. Visual inspection suggests that the two dipole moments are roughly aligned, and electrostatic calculations show that the angle between the dipole vectors is only 30 0 The high affinity of FAS for AChE can be attributed to many residues either unique to FAS or rare in other three-fingered toxins (Giles et al., 1997), and to a remarkable surface complementarity, involving a large contact area (2000 A2). This is substantially larger, for example, than the contact area between lysozyme and an antibody raised against it, 1700 A2, or between trypsin and bovine pancreatic trypsin inhibitor, 1400 A2 (Janin & Chothia, 1990). A most striking and rare interaction is a stacking of the side chains of Met33 in FAS and of Trp279 in Torpedo AChE. Mutation of this tryptophan residue to a nonaromatic residue decreases the affinity of FAS for AChE by over five orders of magnitude (Radic et al., 1994), and its absence from the AChEs cloned so far from avian and invertebrate sources (e.g. Eichler et al., 1994; Cousin et al., 1996), as well as from butyrylcholinesterase BChE (Harel et al., 1992), provides a clear structural explanation for their poor inhibition by FAS.
526 I. Silman et al. CH 3 H "--'"!'7'"""-N Figure 2. Molecular structure of (-)-huperzine A (HupA). HupA-TcACbE Complex (-)-Huperzine A (HupA, Fig. 2) is a nootropic alkaloid extracted from the club moss, Huperzia serrata, which has been used in China for centuries as a folk medicine (Liu et al., 1986). HupA is a potent reversible inhibitor of AChE that lacks potentially complicating muscarinic effects (Kozikowski et al., 1992). The existence of a natural AChE inhibitor, taken together with its unique pharmacological features and relative lack of toxicity (Laganiere et al., 1991), render HupA a particularly promising candidate for treatment of Alzheimer's disease. Indeed, studies on experimental animals reveal significant cognitive enhancement (Xiong et al., 1995), and clinical trials in China have both established the safety of HupA, and provided preliminary evidence for significant effects on patients exhibiting dementia and memory disorders (Zhang et al., 1991). The structure of HupA reveals no obvious similarity to that of ACh. In fact, a number of studies, utilising either computerised docking techniques and/or site-directed mutagenesis (Ashani et al., 1994; Pang et al., 1994; Saxena et al., 1994), predicted various possible orientations of HupA within the active site of AChE. It seemed, therefore, desirable to solve the structure of a TcAChE-HupA complex by X-ray crystallography. It would thus be possible to establish that it indeed binds at the active-site and to determine its correct orientation, thus providing the basis for future structure-based drug design. Soaking of HupA into native trigonal crystals of Torpedo AChE yielded a crystalline complex from which a data set was collected which could be refined to 2.5 A resolution. Examination of a difference map for the complex, as compared to the native enzyme, clearly revealed "a prominent electron density peak near the bottom of the 'aromatic gorge' with an outline resembling that of HupA. Indeed, excellent fitting of the molecule to the electron density was obtained. The crystal structure of the HupA-TcAChE complex (Fig. 3) shows an unexpected orientation for the inhibitor, with surprisingly few strong direct interactions with protein residues to explain its high affinity. The principal interactions include: (a) a strong hydrogen bond (2.6 A) of the carbonyl group of the ligand to Tyr130; (b) hydrogen bonds to water molecules within the active-site gorge which are, themselves, hydrogen-bonded to other waters or to side-chain and backbone atoms of the protein, notably to the carboxylic oxygens of Glu 199 and to the hydroxyl oxygen of Tyr 121; (c) interaction of the primary amino group of the ligand, which can be assume to be charged at the ph of the mother liquor, with the aromatic rings of Trp84 and Phe330; (d) an unusually short (3.0 A) C-H'D bond between the ethylidene methyl group of HupA and the main-chain oxygen of His440; and (e) several hydrophobic contacts notably with the side chains and main-chain atoms of Trp84 and with residues Gly118 through Serl22. Modelling Phe330 in the crystal structure as tyrosine, which is the corresponding residue in mammalian AChE, permits formation of a 3.3 A hydrogen bond between the
Crystallographic Studies on Complexes of Acetylcholinesterase 527 hydroxyl oxygen and the primary amino group of HupA. This extra hydrogen bond, in addition to 1t-cation interactions, may help to explain why HupA binds to mammalian AChE 5-l0-fold more strongly than to TcAChE, and only weakly to BChE, which lacks an aromatic residue at this position (Ashani et al., 1994). It seems surprising that an inhibitor with a relatively high affinity for AChE-K j ca. 6 nm for fetal bovine serum AChE, and 250 nm for TcAChE (Saxena et al., 1994}-binds through so few direct contacts. Even though HupA has three potential hydrogen-bond donor and acceptor sites (Fig. 2), only one strong hydrogen bond is seen, between the pyridone oxygen and Tyrl30. Analogous compounds with a methoxy replacing the oxygen show no inhibition at all (Kozikowski et al., 1992). It is also of interest that the ring nitrogen is hydrogen-bonded to the protein via a water molecule, and hydrogen bonds between the NH 3 + group and the protein are mediated through at least two waters. The aromatic rings of both Trp84 and Phe330 are near the primary amino group. However, the structure displays a large number of hydrophobic interactions: there are 11 contacts between a carbon atom of HupA and oxygen or nitrogen atoms of the protein, and 20 carbon-to-carbon contacts within 4.0 A.. Consequently, there does not appear to be much room for adding additional groups without causing clashes. Nevertheless, addition of a methyl group near the amide group of HupA leads to an 8-fold increase in affinity, probably due to extra hydrophobic contacts with Trp84 (Kozikowski et al., 1996). In summary, the crystal structure of the HupA-TcAChE complex reveals an unexpected orientation of the ligand within the active site, as well as unusual protein-ligand interactions. This information should be of value in the design and analysis of analogs of H:upA with improved pharmacological characteristics. FIgure 3A. Ribbon diagram of the HupA-TcAChE complex, showing the HupA molecule at the bottom of the active-site gorge.
528 I. Silman et al. Figure 38. Enlargement of the active site region. showing the catalytic triad to the right. and some of the aromatic residues surrounding the HupA molecule making contact. ACKNOWLEDGMENTS This research was supported by the U.S. Army Medical Research and Development Command, the Minerva Foundation, the Kimmelman Center for Biomolecular Structure and Assembly, and the Scientific Cooperation of the European Union with Third Mediterranean Countries through the Israeli Ministry of Science. I.S. is Bernstein-Mason Professor of Neurochemistry.
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