Conformational remodeling of femtomolar inhibitor-acetylcholinesterase complexes in the crystalline state

J Am Chem Soc. 2010 Dec 29;132(51):18292-300. doi: 10.1021/ja106820e. Epub 2010 Nov 19.

Abstract

The active center of acetylcholinesterase (AChE), a target site for competitive inhibitors, resides centrosymmetric to the subunit at the base of a deep, narrow gorge lined by aromatic residues. At the gorge entry, a peripheral site encompasses overlapping binding loci for noncompetitive inhibitors, which alter substrate access to the gorge. The click-chemistry inhibitor TZ2PA6 links the active center ligand, tacrine, to the peripheral site ligand, propidium, through a biorthogonal reaction of an acetylene and an azide that forms either a syn1 or an anti1 triazole. Compared with wild-type mouse AChE, a Tyr337Ala mutant displays full catalytic activity, albeit with 2-3 orders of magnitude higher affinities for the TZ2PA6 syn1 and anti1 regioisomers, reflected in low femtomolar K(d) values, diffusion-limited association, and dissociation half-times greater than 1 month and 1 week, respectively. Three structures of each of the co-crystallized syn1 and anti1 complexes of the Tyr337Ala mutant were solved at three distinct times of crystal maturation, consistent with or exceeding the half-lives of the complexes in solution, while crystalline complexes obtained from soaked Tyr337Ala crystals led to picturing "freshly formed" complexes. The structures, at 2.55-2.75 Å resolution, reveal a range of unprecedented conformations of the bound regioisomers, not observed in the wild-type AChE complexes, associated with concerted positional rearrangements of side chains in the enzyme gorge. Moreover, time-dependent conformational remodeling of the crystalline complexes appears to correlate with the dissociation half-times of the solution complexes. Hence, for the tight-binding TZ2PA6 inhibitors, the initial complexes kinetically driven in solution slowly form more stable complexes governed by thermodynamic equilibrium and observable in mature crystals.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylcholinesterase / chemistry*
  • Acetylcholinesterase / genetics
  • Binding, Competitive
  • Catalytic Domain / drug effects
  • Catalytic Domain / genetics
  • Cholinesterase Inhibitors / chemistry*
  • Crystallization
  • Crystallography, X-Ray
  • HEK293 Cells
  • Humans
  • Kinetics
  • Mutation
  • Phenanthridines / chemistry*
  • Protein Conformation
  • Tacrine / analogs & derivatives*
  • Tacrine / chemistry
  • Tyrosine / chemistry
  • Tyrosine / genetics

Substances

  • Cholinesterase Inhibitors
  • Phenanthridines
  • TZ2PA6 compound
  • Tyrosine
  • Tacrine
  • Acetylcholinesterase