INTERNAL WATER MOLECULES IN ACETYLCHOLINESTERASE FROM TORPEDO CALIFORNICA
T. Steiner1, G. Koellner1, I. Silman2 and J. L. Sussman1
1Department of Structural Biology,
2Department of Neurobiology,
Weizmann Institute of Science, Rehovot 76100, Israel
Acetylcholoinesterase (AChE) terminates impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. The active site of the enzyme is located near the bottom of a long and narrow gorge that reaches halfway into the molecule [1]. Several crystal structures of AChE from Torpedo californica are now available with a number of natural and synthetic inhibitors (see Refs. [2,3] and references therein).
Water-biomolecule interactions are of great importance in structural biology, and well-refined water molecule sites in biomolecules are worthy of close attention. In the present study, we analyse the location and the intermolecular contacts of the water molecule sites in four isomorphous TcAChE (TC) crystal structures with resolutions of 2.5 A and better, which have been independently refined. Because of their importance, we concentrate on the internal water molecules (i.e. those having no contact with the bulk solvent) and the water sites within the the active site gorge.
The TcAChE molecule contains 39 internal water molecules, which occur in very similar positions in all four crystal structures, and form identical patterns of intermolecular interactions. Because they are structurally so well conserved, they can be regarded as integral part of the protein. If no substrate or inhibitor is bound, the gorge is filled with about 20 water molecules. Of the 39 internal water molecules, 15 are in isolated positions distributed over the whole molecule. The other 24 are found in 8 larger cavities containing between two and five water molecules each. It is striking that these larger cavities are all located close to the active site gorge and have contact to amino acid residues that are part of the gorge wall. Apparently, the entire intramolecular surrounding of the gorge is not very compactly folded.
Because the internal water molecules are conserved in four crystal structures, relatively detailed analysis can be performed with respect to subtle features of their intermolecular interactions. Several of the water molecules accept C-H...O hydrogen bonds which are conserved in all structures, and can, therefore, with good conscience be regarded as 'true'. One of the water molecules in a larger cavity forms an equally well conserved short contact with the phenyl face of a tryptophane side chain. This is interpreted as an aromatic hydrogen bond of the same kind as the Ow-H...Ph hydrogen bonds recently reported to occur in hydrated peptides [4].