Mia L. Raves

Static Laue Diffraction Studies on Acetylcholinesterase

Acetylcholinesterase (AChE) is one of natures fastest enzymes, despite the fact that its three-dimensional structure reveals its active site to be deeply sequestered within the molecule. This raises questions with respect to traffic of substrate to, and products from, the active site, which may be investigated by time-resolved crystallography. In order to address one aspect of the feasibility of performing time-resolved studies on AChE, a data set has been collected using the Laue technique on a trigonal crystal of Torpedo californica AChE soaked with the reversible inhibitor edrophonium, using a total X-ray exposure time of 24 ms. Electron-density maps obtained from the Laue data, which are of surprisingly good quality compared with similar maps from monochromatic data, show essentially the same features. They clearly reveal the bound ligand, as well as a structural change in the conformation of the active-site Ser200 induced upon binding.

Quaternary Structure of Tetrameric Acetylcholinesterase

Most vertebrates contain a single gene encoding for AChE, and alternative splicing gives rise to two catalytic subunits, H and T. H subunits form GPI-anchored dimers, whereas T subunits can occur as monomers, dimers and tetramers, which are dimers of disulfide-linked dimers. T subunits also associate with structural subunits (P and Q) to form membrane-anchored tetramers and asymmetric forms, in which one to three tetramers are attached to a collagen-like tail (1).

Ab initio structure determination of low-molecular-weight compounds using synchrotron radiation Laue diffraction

The potential of using polychromatic synchrotron radiation for ab initio structure determination of low-molecular-weight compounds is investigated. Three different organic compounds were studied. For each of the structures the cell volume was determined with the aid of a Zr attenuator that was placed in the direct beam to obtain a sharp \lambda_{\rm min} edge. Space-group determination did not prove to be complicated; it was aided by deconvolution of the multiple spots. Two low-temperature data sets were collected, and it appeared that spot streaking owing to the inherent increase of mosaicity due to freezing was not too detrimental to the application of the Laue method. Although both the R_{\rm merge} values (around 12%) and the final R_1 values (around 10%) are higher than usually found in monochromatic experiments, all structures show reasonable geometry. Comparison of one of the structures with a reference structure obtained with monochromatic data shows an r.m.s. deviation between the 21 non-H-atom positions of 0.019 A.

Alternative Crystal Forms of Torpedo Californica Acetylcholinesterase

Proteins often form crystals of different shapes and sizes, depending on crystallisation conditions such as pH, temperature, concentration and nature of precipitant, and the presence or absence of additives. In addition to the macroscopic variety and the quality of diffraction of the crystals, different crystal forms show important differences in the packing of protein molecules. It was noted by Axelsen et al. [1] that, due to crystal contacts, the entrance to the active-site gorge of every monomer in the trigonal crystal form of Torpedo californica acetyl-cholinesterase (TcAChE) is tightly blocked by a symmetry-related molecule.

Crystal Structures of “Aged” Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase

Organophosphates (OP) are potent transition state (TS) inhibitors which react rapidly with acetylcholinesterase (AChE), and then may undergo an internal dealkylation to produce an irreversibly inhibited, “aged” OP-enzyme conjugate. To understand the structural basis for the stability of aged enzyme, we crystallized and solved the X-ray structures of conjugates obtained by reaction of Torpedo californica (Tc) AChE with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman). After reaction with OP, unbound inhibitor was removed by gel filtration and aging was allowed to proceed to >90% completion. Aged OP-TcAChE was crystallized using PEG-200 and MES buffer at pH 5.8. X-ray data were collected using trigonal crystals, and refined using difference Fourier techniques at 2.2A (DFP), 2.5A (sarin), and 2.2A (soman) resolution. In each structure, the highest positive difference density peak, corresponding to the OP, was observed to be within covalent bonding distance of Ser200. All three structures suggest that the stability of aged AChE derives from interaction of the two resonance oxygen atoms attached to the phosphorus atom with catalytic subsites of the enzyme. Based upon the geometry of the refined structures, we infer that backbone amides of the oxyanion hole (Gly118, Gly119 and Ala201) stabilize one oxygen by hydrogen bonding, while the His440 imidazolium holds the other oxygen in a salt bridge. The conformations of the active sites of aged sarin- and soman-TcAChE are essentially identical and provide structural models for the rate-limiting deacylation TS that occurs during enzyme hydrolysis of the natural substrate, acetylcholine.

Crystallographic Studies on Complexes of Acetylcholinesterase with the Natural Cholinesterase Inhibitors Fasciculin and Huperzine A

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 acive 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.

How Three-Fingered Snake Toxins Recognise Their Targets

Three-fingered toxins from snake venoms constitute a family of 6-8kDa proteins, which can be divided into a number of groups with widely varying targets. The most studied of these are the α-neurotoxins which have long been used for the purification and characterisation of nicotinic acetylcholine receptors. Another group are the fasciculins, which specifically inhibit another essential synaptic component, the enzyme acetylcholi-nesterase.

Activity of Torpedo Californica Acetylcholinesterase in the Crystalline State

Numerous studies have demonstrated that enzymes display catalytic activity in the crystalline state [1]. Factors which might affect activity of a crystalline enzyme include: i. Ability of substrates to reach the active site and of products to exit ii. Chemical composition of the crystallisation mother liquor (ML) iii. Conformational freedom of residues involved in substrate binding and hydrolysis

Structures of Complexes of Acetylcholinesterase with Covalently and Non-Covalently Bound Inhibitors

The principal biological role of acetylcholinesterase (AChE, acetylcholine hydrolase, EC 3.1.1.7) is to terminate signal transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine (ACh) (Barnard, 1974). In keeping with this requirement, AChE possesses a remarkably high specific activity, especially for a serine hydrolase (Quinn, 1987), functioning at a rate approaching that of a diffusion-controlled reaction (Bazelyansky et al., 1986). Early kinetic studies indicated that the active site of AChE consists of two subsites, the ‘esteratic’ and ‘anionic’ subsites, corresponding to the catalytic machinery and the choline-binding pocket, respectively (Nachmansohn and Wilson, 1951). A second, ‘peripheral’, anionic site exists, so named because it appears to be distant from the active site (Taylor and Lappi, 1975). The elucidation of the three-dimensional structure of Torpedo AChE (Sussman et al., 1991) served to confirm these earlier studies, and has showed that AChE contains a catalytic triad similar to that present in other serine hydrolases (Steitz and Shulman, 1982). Unexpectedly, it also revealed that this triad is located near the bottom of a deep and narrow cavity, ∼20 A deep, which has been named the ‘active-site gorge’. The cavity is lined by the rings of fourteen aromatic residues which are conserved in the AChE sequences published so far (Gentry and Doctor, 1991). Much of the subsequent research on structure-function relationships in AChE has been concerned with the functional significance of the gorge and with the role of the aromatic rings which account for more than 50% of its surface area (Axelsen et al., 1994). Thus, structural evidence (Axelsen et al., 1994; Sussman et al., 1991), as well as evidence obtained by modeling (Harel et al., 1992), by chemical modification (Harel et al., 1993; Schalk et al., 1992; Weise et al., 1990), and by site-directed mutagenesis (Harel et al., 1992; Ordentlich et al., 1993; Radie et al., 1993; Shafferman et al., 1992; Vellom et al., 1993), all point to important roles for certain of these conserved aromatic residues in both the ‘esteratic’ and ‘anionic’ subsites of the active site, and in the ‘peripheral’ anionic site.

3-D Structure of Acetylcholinesterase and Its Complexes with Anticholinesterase Agents

The principal biological role of acetylcholinesterase (AChE) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine (ACh). Based on our recent X-ray crystallographic structure determination of AChE from Torpedo californica we can see, at atomic resolution, a protein binding pocket for the neurotransmitter ACh. We found that the active site consists of a catalytic triad (Ser200-His440-Glu327) which lies close to the bottom of a deep and narrow gorge, that is lined with the rings of 14 aromatic amino acid residues. Despite the complexity of this array of aromatic rings, we suggested, on the basis of modelling which involved docking of the ACh molecule in an all-trans conformation, that the quaternary group of the choline moiety makes close contact with the indole ring of Trp84. In order to study experimentally this interaction, in detail, we soaked into crystals of AChE a series of different inhibitors, including the competitive inhibitor edrophonium (EDR) and the transition-state analog (N,N,N-trimethylammonio) trifluoroacetophenone (TFK), and determined their 3-D structures.