Journal:Protein Science:1

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

Aviv Paz, Esther Roth, Yacov Ashani, Yechun Xu, Valery L. Shnyrov, Joel L. Sussman, Israel Silman, and Lev Weiner^[1]

Molecular Tour
The photosensitizer, methylene blue (MB) (colored in darkmagenta), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. The TcAChE active site consists of two binding subsites. One of them is the "catalytic anionic site" (CAS), which involves the catalytic triad Ser200, His440, and Glu327 (colored in orange) and the conserved residues Trp84 and Phe330 which also participate in ligand recognition. Another conserved residue Trp279 (colored in cyan) is situated at the second binding subsite, termed the "peripheral anionic site" (PAS), ~14 Å from CAS. Thioflavin T (2j3q) is a good example of the PAS-binding AChE inhibitors. Superposition of the structure of known CAS-binding inhibitor edrophonium/TcAChE (2ack) on the thioflavin T/TcAChE complex structure (2j3q) shows that these ligands' positions do not overlap^{[2] [3]}. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both ‘‘anionic’’ subsites, but a single site is involved in inhibition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS (2w9i); it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. Superposition of the PAS regions of the MB/TcAChE (2w9i) and thioflavin T/TcAChE (2j3q) complexes reveals similarity between positions of these ligands. As the conformation of TcAChE in the crystal structures of the two complexes is practically identical, only that of the MB/TcAChE structure (2w9i) is shown. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS (2nd MB is colored blueviolet, Phe330 of the crystal structure is in orange and Phe330 of the modeled structure is in indigo). Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.