(-)-Huperzine A (HupA) is found in an extract from a club moss that has been used for centuries in Chinese folk medicine. Its action has been attributed to its ability to strongly inhibit acetylcholinesterase (AChE). The crystal structure of the complex of AChE with optically pure HupA at 2.5 A resolution shows an unexpected orientation for the inhibitor with surprisingly few strong direct interactions with protein residues to explain its high affinity. This structure is compared to the native structure of AChE devoid of any inhibitor as determined to the same resolution. An analysis of the affinities of structural analogues of HupA, correlated with their interactions with the protein, shows the importance of individual hydrophobic interactions between HupA and aromatic residues in the active-site gorge of AChE.

Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.

Chemical modification of Torpedo californica acetylcholinesterase by various sulfhydryl reagents results in its conversion to one of two principal states. One of these states, viz., that produced by disulfides and alkylating agents, is stable. The second state, produced by mercury derivatives, is metastable. At room temperature, it converts spontaneously, with a half-life of ca. 1 h, to a stable state similar to that produced by the disulfides and alkylating agents. Demodification of acetylcholinesterase freshly modified by mercurials, by its exposure to reduced glutathione, causes rapid release of the bound mercurial, with concomitant recovery of most of the enzymic activity of the native enzyme. In contrast, similar demodification of acetylcholinesterase modified by disulfides yields no detectable recovery of enzymic activity. Spectroscopic measurements, employing CD, intrinsic fluorescence, and binding of 1-anilino-8-naphthalenesulfonate, show that the state produced initially by mercurials is "native-like", whereas that produced by disulfides and alkylating agents, and after prolonged incubation of the mercurial-modified enzyme, is partially unfolded and displays many of the features of the "molten globule" state. Arrhenius plots show that the quasi-native state produced by organomercurials is separated by a low (5 kcal/mol) energy barrier from the native state, whereas the partially unfolded state is separated from the quasi-native state by a high energy barrier (ca. 50 kcal/mol). Comparison of the 3D structures of native acetylcholinesterase and of a heavy-atom derivative obtained with HgAc2 suggests that the mercurial-modified enzyme may be stabilized by additional interactions of the mercury atom attached to the free thiol group of Cys231, specifically with Ser228O gamma with the main-chain nitrogen and carbonyl oxygen of the same serine residue.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculinmAChE complex. Complexes of two distinct fasciculins, Fasl-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6,22 or P6,22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a V,, value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of FasZAChE complex (72 kDa) per asymmetric unit. The complex Fasl-mAChE crystallizes in the same space group with identical cell dimensions.