SNAP-25, an intrinsically disordered protein, assembles with syntaxin and synaptobrevin to form a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, a four-helix bundle that plays a key role in the exocytosis of synaptic vesicles. Botulinum neurotoxins specifically cleave one of the SNARE proteins, resulting in the inhibition of membrane fusion. Small molecule inhibitors are being evaluated as potential therapeutic counter-measurement to the toxin's deleterious action on the neuromuscular junctions.
In order to understand the conformational changes exhibited by SNAP-25 prior to and during SNARE complex formation, and its biological function, we developed a method of investigating the conformational changes of SNAP-25 in the absence and presence of Zn2+-depleted BoNT/A LC (botulinum neurotoxin A light chain), using FRET (fluorescence resonance energy transfer) between EGFP (enhanced green fluorescent protein) and CCPGCC-ReAsH fused to SNAP-25 at specific locations. The apparent distances between the C-terminus and the N-terminus, the C-terminus and the D140, and the N-terminus and the D140 of the SNAP-25 protein were determined based on the FRET efficiency in the absence and presence of Zn2+-depleted BoNT/A LC. Various mutants of SNAP-25(A195S) were genetically engineered and used to measure the tertiary structures of SNAP-25(A195S) in the absence and presence of active BoNT/A LC. Shorter apparent distances in the presence of BoNT/A LC suggest that the SNAP-25 folds around BoNT/A LC in the binary complex. FRET is a useful tool to investigate the conformational changes of other intrinsically disordered proteins.
The secondary structures of the full length SNAP-25(1-206), the N-terminal SNAP-25(1-140), and the C-terminal SNAP-25(141-206) and their corresponding N-terminal CCPGCC modified fragments were measured by CD. The results suggest that SNAP-25 and its N-terminal and C-terminal domains are intrinsically disordered proteins. Higher á-helical content was observed when the N-terminal and the C-terminal domains of the SNAP-25 bound to BoNT/A LC. We found that not only the C-terminal domain of SNAP-25(141-206), but also the N-terminal domain of SNAP-25(1-140) specifically bound to BoNT/A LC by means of a slow and tight binding.
Quinolinol derivatives were found to be effective inhibitors of botulinum neurotoxin serotype A (BoNT/A). Studies of the inhibitors and binding of 7-phenyl(8-quinolinylamino)-8-quinolinol (QAQ) to BoNT/A LC demonstrated that QAQ is a non-competitive inhibitor of zinc protease activity. Binding and molecular modeling studies revealed that QAQ binds to a hydrophobic pocket near the active site. A 24-mer SNAP-25 peptide, containing E183 to G206, with a Q197C mutation (Peptide C), binds to BoNT/A LC with an unusually slow second order binding rate constant of 76.7 M -1·s-1. QAQ binds to Zn2+-depleted BoNT/A LC with a KD of 0.67 µM and to the Peptide C-BoNT/A LC complex with a KD of 2.33 µM. Insights into the interactions between the quinolinols and peptides and the zinc protease of BoNT/A should aid in the development of metalloprotease inhibitors.
|Advisor:||Yang, David C. H., Wolf, Christian|
|Commitee:||Metallo, Steven J., Smith, Leonard A.|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 72/01, Dissertation Abstracts International|
|Keywords:||Botulinum neurotoxin, FRET, Inhibitors, Membrane fusion, Proteases, Quinolinols|
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