Structural mechanisms and insights on multiple nanobodies binding diverse SOD1 epitopes

Area of Science:

• Biochemistry
• Structural Biology
• Neuroscience

Background:

• Copper/zinc superoxide dismutase (SOD1) is vital for mitigating oxidative stress.
• SOD1 mutations and aggregation are implicated in amyotrophic lateral sclerosis (ALS) pathogenesis.
• Nanobodies offer a targeted therapeutic strategy for pathogenic SOD1.

Purpose of the Study:

• To determine the high-resolution crystal structures of SOD1 in complex with nanobodies.
• To characterize the binding affinities and interactions of single and multimeric nanobodies with SOD1.
• To establish a foundation for developing nanobody-based tools for ALS.

Main Methods:

• X-ray crystallography to determine complex structures.
• Isothermal titration calorimetry (ITC) and fluorescence-detection size-exclusion chromatography (FSEC) for binding affinity.
• Dynamic light scattering (DLS) for complex stability.

Main Results:

• First high-resolution crystal structures of SOD1-nanobody complexes (1:2 and 1:3 stoichiometries).
• Nanobodies bind SOD1 with nanomolar affinities (K_D 23.2 nM to 529 nM).
• Engineered tandem nanobodies achieved higher affinity (K_D 4.39 nM) without steric interference.

Conclusions:

• Atomic-resolution insights into multi-nanobody targeting of SOD1.
• Demonstrated high-affinity binding and colloidal stability of SOD1-nanobody complexes.
• Provides a basis for developing SOD1-targeting tools for ALS and neurodegenerative diseases.