Gitay Kryger

Structure of acetylcholinesterase complexed with (-)-galanthamine at 2.3 A resolution.

(−)‐Galanthamine (GAL), an alkaloid from the flower, the common snowdrop (Galanthus nivalis), shows anticholinesterase activity. This property has made GAL the target of research as to its effectiveness in the treatment of Alzheimers disease. We have solved the X‐ray crystal structure of GAL bound in the active site of Torpedo californica acetylcholinesterase (TcAChE) to 2.3 Å resolution. The inhibitor binds at the base of the active site gorge of TcAChE, interacting with both the choline‐binding site (Trp‐84) and the acyl‐binding pocket (Phe‐288, Phe‐290). The tertiary amine group of GAL does not interact closely with Trp‐84; rather, the double bond of its cyclohexene ring stacks against the indole ring. The tertiary amine appears to make a non‐conventional hydrogen bond, via its N‐methyl group, to Asp‐72, near the top of the gorge. The hydroxyl group of the inhibitor makes a strong hydrogen bond (2.7 Å) with Glu‐199. The relatively tight binding of GAL to TcAChE appears to arise from a number of moderate to weak interactions with the protein, coupled to a low entropy cost for binding due to the rigid nature of the inhibitor.

Solvent behaviour in flash-cooled protein crystals at cryogenic temperatures

The solvent behaviour of flash-cooled protein crystals was studied in the range 100--180 K by X-ray diffraction. If the solvent is within large channels it crystallizes at 155 K, as identified by a sharp change in the increase of unit-cell volume upon temperature increase. In contrast, if a similar amount of solvent is confined to narrow channels and/or individual cavities it does not crystallize in the studied temperature range. It is concluded that the solvent in large channels behaves similarly to bulk water, whereas when confined to narrow channels it is mainly protein-associated. The analogy with the behaviour of pure bulk water provides circumstantial evidence that only solvent in large channels undergoes a glass transition in the 100--180 K temperature range. These studies reveal that flash-cooled protein crystals are arrested in a metastable state up to at least 155 K, thus providing an upper temperature limit for their storage and handling. The results are pertinent to the development of rational crystal annealing procedures and to the study of temperature-dependent radiation damage to proteins. Furthermore, they suggest an experimental paradigm for studying the correlation between solvent behaviour, protein dynamics and protein function.

Towards Atomic Resolution of Prokaryotic Ribosomes: Crystallographic, Genetic and Biochemical Studies

The studies reported here were initiated and inspired by the late Prof. H.G. Wittmann. From the early stages of this project, when it was widely believed that even the initial steps in determining the molecular structure of ribosomes are impossible, until his last days, Prof. Wittmann was actively involved in the experimental design and in the actual studies. We have no doubt that without his motivation, optimism, guidance and support, this project would not have reached its current stage.

3D Structure at 2.7 Å Resolution of Native and E202Q Mutant Human Acetylcholinesterase Complexed with Fasciculin-II

Knowledge of the 3D structure of human acetylcholinesterase (AChE) is of importance for drug design, in particular of anti-Alzheimer drugs (1, 2), for development of improved procedures for treatment of intoxication by nerve agents (3), and for the design of safer and more effective insecticides.

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.

3D Structure of a Complex of Human Acetylcholinesterase with Fasciculin-II at 2.7 Å Resolution

A knowledge of the 3D structure of human acetylcholinesterase (AChE) is of importance for the development of anti-Alzheimer drugs, for the general understanding of organophosphate toxicity and for the design of safer and more specific insecticides. A mutant of recombinant human acetylcholinesterase (rhAChE) was constructed in which the C-terminus had been truncated to give rise to a homogeneous monomeric form. The monomeric rhAChE was expressed in HEK 293 cells and purified by affinity chromatog-raphy. The purified enzyme was co-crystallized from ammonium sulfate at pH 7.2 and 19°C as a stoichiometric complex with the mamba venom polypeptide toxin, fasciculin-II (FAS-II). X-ray data were collected at the BNL-NSLS X12-C source from a single cryogenically cooled crystal. The overall completeness of the 30A-2.7A data is 99.0% and the R-merge is 8.1%. The model was refined starting from the mouse AChE-FAS-II structure to Rfree of 29% and R of 21.9% at 2.7A resolution. The overall fold is similar to that of Torpedo californica and mouse complexes with FAS-II. A mutant E202(199)Q was prepared and crystallized in a similar fashion. Although significantly different from the native enzyme in activity, the E202(199)Q is almost identical to the native in its 3D structure.

3D Structure of a Complex of the Anti-Alzheimer Drug, E2020, with Acetylcholinesterase at 2.5Å Resolution

Observations documenting adverse effects of anticholinergic drugs on memory, taken together with postmortem data which revealed low cholinergic activities in Alzheimer’s Disease (AD) patients, led to the hypothesis, known as the ‘cholinergic hypothesis’, that AD is associated with an impairment in cholinergic transmission (1). This led to the suggestion that cholinesterase (ChE) inhibitors would reverse deficits in acetylcholine (ACh) levels associated with AD, and thus might reverse the memory impairments characteristic of the disease. Consequently, a number of ChE inhibitors have been considered as candidates for the symptomatic treatment of AD, and have been utilized in clinical trials. They include natural substances, such as physostigmine (2) and huperzine A (3), both of which are alkaloids, and synthetic compounds, such as SDZ ENA-713, also known as Exelon® (4), and metrifonate (5). Recently, evidence was presented that AChE may contribute to the generation of amyloid proteins and/or physically affect the process of fibril assembly which results in the formation of the senile plaques characteristic of AD (6). It was suggested that a hydrophobic environment close to the peripheral binding site of the enzyme, at or near the entrance to the active-site gorge, may be involved in this process (7).

Crystal Structures of Complexes of E2020-Related Compounds with Torpedo Californica Acetylcholinesterase

E2020 is a potent inhibitor of acetylcholinesterase (AChE), displaying high selectivity for AChE relative to butyrylcholinesterase. It was recently licensed under the trade name of Aricept by the FDA for use in the management of Alzheimer’s disease. A series of compounds related to E2020 were synthesized and shown to serve as good inhibitors of Torpedo californica acetylcholinesterase (TcAChE). Several of these inhibitors were soaked into TcAChE crystals, and the three-dimensional structures of the complexes obtained were solved by X-ray crystallography. As might be expected from their elongated structures the ligands bind along the axis of the active-site gorge, interacting with the peripheral anionic site (Trp279), as well as with various other aromatic groups along the length of the gorge. The benzyl-piperidine group binds in the area of Trp84, the residue which binds the quaternary ammonium cation of acetylcholine. Inhibitors in which a trifluoroketo group had been added to the phenyl ring formed a covalent bond with the Oγ of the active site serine, Ser200. It is hoped that these studies will clarify which interactions are most important for tight inhibitor binding, and thus permit the design of more effective and selective insecticides which will simultaneously be acutely toxic for their insect targets and relatively harmless to other animals.