Structural dynamics of the alpha-neurotoxin-acetylcholine-binding protein complex: hydrodynamic and fluorescence anisotropy decay analyses.
The three-fingered alpha-neurotoxins have played a pivotal role in elucidating the structure and function of the muscle-type and neuronal alpha7 nicotinic acetylcholine receptors (nAChRs). To advance our understanding of the alpha-neurotoxin-nAChR interaction, we examined the flexibility of alpha-neurotoxin bound to the acetylcholine-binding protein (AChBP), which shares structural similarity and sequence identities with the extracellular domain of nAChRs. Because the crystal structure of five alpha-cobratoxin molecules bound to AChBP shows the toxins projecting radially like propeller "blades" from the perimeter of the donut-shaped AChBP, the toxin molecules should increase the frictional resistance and thereby alter the hydrodynamic properties of the complex. alpha-Bungarotoxin binding had little effect on the frictional coefficients of AChBP measured by analytical ultracentrifugation, suggesting that the bound toxins are flexible. To support this conclusion, we measured the anisotropy decay of four site-specifically labeled alpha-cobratoxins (conjugated at positions Lys(23), Lys(35), Lys(49), and Lys(69)) bound to AChBP and free in solution and compared their anisotropy decay properties with fluorescently labeled cysteine mutants of AChBP. The results indicated that the core of the toxin molecule is relatively flexible when bound to AChBP. When hydrodynamic and anisotropy decay analyses are taken together, they establish that only one face of the second loop of the alpha-neurotoxin is immobilized significantly by its binding. The results indicate that bound alpha-neurotoxin is not rigidly oriented on the surface of AChBP but rather exhibits segmental motion by virtue of flexibility in its fingerlike structure.
Hibbs
Ryan E
RE
Department of Pharmacology and Biomedical Sciences Graduate Program, University of California-San Diego, La Jolla, California 92093-0636, USA.
Johnson
David A
DA
Shi
Jianxin
J
Hansen
Scott B
SB
Taylor
Palmer
P
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D009498:Neurotoxins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism; D014461:Ultracentrifugation; D064569:alpha7 Nicotinic Acetylcholine Receptor
C428944:AChBP protein, Lymnaea; D002352:Carrier Proteins; C579167:Chrna7 protein, human; D005456:Fluorescent Dyes; D009498:Neurotoxins; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor