Structural basis for high-affinity actin binding revealed by a β-III-spectrin SCA5 missense mutation

Area of Science:

• Biochemistry
• Structural Biology
• Neuroscience

Background:

• Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disorder.
• It results from mutations in the cytoskeletal protein β-III-spectrin.
• A known SCA5 mutation (L253P) in the actin-binding domain (ABD) increases actin-binding affinity 1000-fold.

Purpose of the Study:

• To elucidate the structural basis for the increased actin-binding affinity caused by the L253P mutation in β-III-spectrin's ABD.
• To understand how this mutation contributes to the pathogenesis of SCA5.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of F-actin complexed with the L253P ABD at 6.9 Å resolution.
• Co-sedimentation assays to measure binding affinity.
• Pulsed electron paramagnetic resonance (pulsed-EPR) spectroscopy to probe conformational changes.

Main Results:

• The cryo-EM structure revealed that the L253P mutation causes the two CH domains to open.
• This opening facilitates binding of the CH1 domain to actin.
• An N-terminal region, initially unstructured, becomes α-helical upon binding and is crucial for this interaction.

Conclusions:

• The L253P mutation enhances β-III-spectrin's actin-binding affinity by altering domain conformation and engaging an N-terminal helix.
• This provides insight into the molecular mechanisms of actin-binding proteins and how mutations cause neurodegenerative diseases like SCA5.