Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly.

Area of Science:

• Molecular Biology
• Neuroscience
• Biophysics

Background:

• Synaptic exocytosis involves SNARE proteins forming a four-helix bundle to drive membrane fusion.
• SNARE assembly occurs via stepwise zippering, with C-terminal zippering acting as the power stroke.
• SNARE mutations are linked to neurological disorders, but their precise impact on assembly energetics is unclear.

Purpose of the Study:

• To quantify the energetic and kinetic effects of specific SNAP-25B mutations (I67T/N) on SNARE complex assembly.
• To investigate how these mutations influence the zippering process and the stability of the target SNARE complex.
• To elucidate the molecular mechanisms underlying neurological disorders associated with altered SNARE function.

Main Methods:

• Utilized single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes.
• Introduced single mutations I67T/N into the neuronal SNARE synaptosomal-associated protein of 25kDa (SNAP-25B).
• Analyzed the impact of mutations on N-terminal and C-terminal zippering, as well as the structural stability of the t-SNARE complex.

Main Results:

• Both I67T/N mutations significantly reduced the energy of C-terminal SNARE zippering by approximately 10 k_BT.
• N-terminal SNARE assembly was not affected by these mutations.
• Mutations were observed to cause unfolding of the C-terminal region within the t-SNARE complex.

Conclusions:

• SNAP-25B mutations I67T/N impair synaptic exocytosis by destabilizing SNARE complex assembly.
• The findings contradict previous hypotheses suggesting stabilization of SNARE assembly.
• This study provides critical insights into the molecular mechanisms of neurological diseases caused by SNARE mutations.