Structure of acetylcholinesterase complexed with the nootropic alkaloid, (–)-huperzine A

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The X-ray crystal structure of Torpedo californica acetylcholinesterase (TcAChE) complexed with BW284C51 {CO[ÐCH 2 CH 2 ÐpC 6 H 4 ÐN(CH 3 ) 2 (CH 2 ÐCH CH 2 )] 2 } is described and compared with the complexes of two other active-site gorge-spanning inhibitors, decamethonium and E2020. The inhibitor was soaked into TcAChE crystals in the trigonal space group P3 1 21, yielding a complex which diffracted to 2.85 A Ê resolution. The structure was re®ned to an R factor of 19.0% and an R free of 23.4%; the ®nal model contains the protein, inhibitor, 132 water molecules and three carbohydrate moieties. BW284C51 binds similarly to decamethonium and E2020, with its two phenyl and quaternary amino end-groups complexed to Trp84 in the catalytic site and to Trp279 in the peripheral binding site, and its central carbonyl group hydrogen bonded very weakly to Tyr121. Possible reasons for decamethonium's weaker binding are considered. The relative strength of binding of bisquaternary inhibitors to acetylcholinesterase and the effect of several mutations of the enzyme are discussed in the context of the respective X-ray structures of their complexes with the enzyme.

Section: Figurementioning

confidence: 99%

Structure of a complex of the potent and specific inhibitor BW284C51 withTorpedo californicaacetylcholinesterase

Felder

Harel

Silman

et al. 2002

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“…Two crystal forms of TcAChE have been studied: trigonal crystals (Sussman et al, 1991;Raves et al, 1997) containing 68% solvent arranged in large solvent channels and orthorhombic crystals containing 56% solvent accommodated in individual cavities and narrow channels (Raves, 1998). 2gpI crystals have an unusually high solvent content of 86% and possess even larger solvent channels than trigonal TcAChE (Bouma et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Solvent behaviour in flash-cooled protein crystals at cryogenic temperatures

Weik

Kryger

Schreurs

et al. 2001

The solvent behaviour of¯ash-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 identi®ed by a sharp change in the increase of unit-cell volume upon temperature increase. In contrast, if a similar amount of solvent is con®ned 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 con®ned 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¯ash-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.

“…The structures of native T. californica acetylcholinesterase (TcAChE; Sussman et al, 1991) and of various complexes and conjugates with, respectively, reversible and covalent inhibitors Harel et al, 1993;Kryger et al, 1999;Millard et al, 1999;Raves et al, 1997) have been solved by X-ray crystallography. Even though many insights into key structural elements of the catalytic and anionic binding sites were obtained, the localization of the binding sites for divalent ions such as magnesium, calcium and manganese would help to elucidate their putative structural and functional roles.…”

Section: Introductionmentioning

confidence: 99%

Histochemical method for characterization of enzyme crystals: application to crystals ofTorpedo californicaacetylcholinesterase

Nicolas

Ferrón

Toker

et al. 2001

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Histochemical methods are employed to detect and localize a wide range of enzymes. Even though protein crystallographers do not commonly use this technique, the extensively used colorimetric reaction of Karnovsky was successfully adapted for easy and quick identi®cation of acetylcholinesterase crystals. The method relies on the reduction of ferricyanide to ferrocyanide by thiocholine, released from acetylthiocholine by enzymatic hydrolysis, followed by formation of a cupric ferrocyanide precipitate, and allows rapid differentiation between salt and enzyme crystals and between native and inhibited crystals of the enzyme.