Crystal structures of a cysteine-modified mutant in loop D of acetylcholine-binding protein.
Covalent modification of α7 W55C nicotinic acetylcholine receptors (nAChR) with the cysteine-modifying reagent [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET(+)) produces receptors that are unresponsive to acetylcholine, whereas methyl methanethiolsulfonate (MMTS) produces enhanced acetylcholine-gated currents. Here, we investigate structural changes that underlie the opposite effects of MTSET(+) and MMTS using acetylcholine-binding protein (AChBP), a homolog of the extracellular domain of the nAChR. Crystal structures of Y53C AChBP show that MTSET(+)-modification stabilizes loop C in an extended conformation that resembles the antagonist-bound state, which parallels our observation that MTSET(+) produces unresponsive W55C nAChRs. The MMTS-modified mutant in complex with acetylcholine is characterized by a contracted C-loop, similar to other agonist-bound complexes. Surprisingly, we find two acetylcholine molecules bound in the ligand-binding site, which might explain the potentiating effect of MMTS modification in W55C nAChRs. Unexpectedly, we observed in the MMTS-Y53C structure that ten phosphate ions arranged in two rings at adjacent sites are bound in the vestibule of AChBP. We mutated homologous residues in the vestibule of α1 GlyR and observed a reduction in the single channel conductance, suggesting a role of this site in ion permeation. Taken together, our results demonstrate that targeted modification of a conserved aromatic residue in loop D is sufficient for a conformational switch of AChBP and that a defined region in the vestibule of the extracellular domain contributes to ion conduction in anion-selective Cys-loop receptors.
Laboratory of Structural Neurobiology, KULeuven, 3000 Leuven, Belgium.
Marijke
M
Brams
Elaine A
EA
Gay
José Colón
JC
Sáez
Albert
A
Guskov
René
R
van Elk
Roel C
RC
van der Schors
Steve
S
Peigneur
Jan
J
Tytgat
Sergei V
SV
Strelkov
August B
AB
Smit
Jerrel L
JL
Yakel
Chris
C
Ulens
D000109:Acetylcholine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001048:Aplysia / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D002352:Carrier Proteins; D003545:Cysteine; D000109:Acetylcholine