3-D Structural Studies of Proteins and Nucleic Acids

3-D Structures of Acetylcholinesterase Complexes. Following the determination of the 3-D structure of the native acetylcholinesterase (AChE), we studied its unique properties as a key enzyme in the nervous system by determining the structures of complexes of it with a series of inhibitors. Structures of AChE complexed with very potent bisquaternary inhibitors, decamethonium and BW251, were solved showing one of their quaternary groups interacting with the active site TRP-84, and the other interacting with TRP-279 at the entrance to the gorge, thus pointing to the location of the 'peripheral' birding site of AChE, and a complex with 2-PAM (an antidote to insecticide and nerve gas poisoning) was solved, showing its interactions in the active site. The transition state analog, TMTFA, was solved showing a covalently bound inhibitor and thus indicating how the natural substrate, acetylcholine, binds to AChE. Structures to higher resolutions (2.25 A compared with 2.8 A), based on data collected at the NSLS beam line X12c, were solved, revealing missing side-chains and two additional residues not seen at lower resolution. A model of butyryl cholinesterase was built by mutating the active-site gorge residues of AChE, indicating mutating three residues in AChE would produce an enzyme with properties of butyryl cholinesterase. We confirmed this prediction by experiments using site-directed mutagensis.

The 3-D structure of a complex between Torpedo californica, AChE, and the three-fingered snake toxin fasciculin-ll (FAS-II), from the venom of the green mamba, Dendroaspis angusticeps, was determined to 3.0 A resolution. The structure reveals a stochiometric complex with one FAS bound to each AChE subunit. FAS is bound at the 'peripheral' anionic site of AChE, sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of FAS for AChE is due to a remarkable surface complementarily, involving a large contact area, cat 2000 A, and many residues either unique to FAS or rare in other three-fingered toxins. Several basic residues in FAS make important contacts with AChE. The complementarity between FAS and AChE is unusual, inasmuch as it involves a number of charged residues, but lacks any inter-protein salt linkages.

Interactive 3-D Browser for Searching the Protein Data Bank. An X-windows-based interactive browser for searching the ca 4,000 three-dimensional structures of biological macromolecules in the Protein Data Bank (PDB) was developed. It greatly enhances the PDB's printed index listings and various ad-hoc search protocols which have been developed for finding PDB entries. Its highly modular and flexible design allows for rapid modification to meet changing needs.

Selected Publications

Gilson, M.K., Straatsma, T.P., McCammon, J.A., Ripoll, D.R., Faerman, C.H., Axelsen, P., Silman, L, and Sussman, J.L. (1994). Open "back door" in a Molecular Dynamics Simulation of Acetylcholinesterase. Science 263: 1276-1278.

Kreimer, D.l., Dolginiva, E A., Raves, M., Sussman, J.L., Silman, l., and Weiner, L. (1994). A Metastable State of Torpedo Acetylcholinesterase generated by modification with organomercurials. Biochemistry 33: 14407-14418.

Harel, M., Kleywegt, G.J., Ravelli, R.B.G., Silman, l., and Sussman, J.L. (1995). Crystal structure of an acetylcholinesterase-fasciculin complex:interaction of a three-fingered toxin from snake venom with its target. Structure 3: 1355-1366.

Dym, O., Mevarech, M., and Sussman, J.L. (1995). Structural features that stabilize halophilic malate dehydrogenase from an archaebacterium. Science 267: 1344-1346.

Stampf, D.R., Eelder, C.E., and Sussman, J.L. (1995). PDBBrowse - A graphics interface to the Brookhaven Protein Data Bank. Nature 374: 572-574.