The binding site of acetylcholine receptor: from synthetic peptides to solution and crystal structure.
Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) alpha-subunit for the analysis of the binding site of the AChR. A 13-mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by alpha-bungarotoxin (alpha-BTX) from a phage-display peptide library. The solution structure of a complex between this library-lead peptide and alpha-BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure-function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to alpha-BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high-affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of alpha-BTX to the AChR with an IC(50) of 2 nM. The solution and crystal structures of complexes of alpha-BTX with HAP were solved, demonstrating that the HAP fits snugly to alpha-BTX and adopts a beta-hairpin conformation. The X-ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that alpha-BTX wraps around the receptor binding-site loop and, in addition, binds tightly at the interface of two of the receptor subunits, where it inserts a finger into the ligand-binding site. Our proposed model explains the strong antagonistic activity of alpha-BTX and accommodates much of the biochemical data on the mode of interaction of alpha-BTX with the AChR.
Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. sara.fuchs@weizmann.ac.il
Sara
S
Fuchs
Roni
R
Kasher
Moshe
M
Balass
Tali
T
Scherf
Mchal
M
Harel
Mati
M
Fridkin
Joel L
JL
Sussman
Ephraim
E
Katchalski-Katzir
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002038:Bungarotoxins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D019151:Peptide Library; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
D002038:Bungarotoxins; D019151:Peptide Library; D010455:Peptides; D011950:Receptors, Cholinergic