A Few Charged Residues in Galectin-3's Folded and Disordered Regions Regulate Phase Separation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Proteins with intrinsically disordered regions (IDRs) regulate cellular functions via phase separation.
• Lysosomal damage causes abrupt pH changes, impacting protein behavior.
• The response of protein phase separation to varying pH is poorly understood.

Purpose of the Study:

• To investigate how pH influences the phase separation of proteins, specifically galectin-3.
• To elucidate the molecular interactions driving galectin-3 assembly and its pH sensitivity.

Main Methods:

• Extensive mutagenesis to alter protein sequences.
• Nuclear Magnetic Resonance (NMR) spectroscopy to study protein structure and dynamics.
• Biophysical techniques to assess phase separation and binding interactions.

Main Results:

• Galectin-3 assembly is driven by cation-π interactions between its folded domain and IDR, alongside π-π interactions within IDRs.
• Two specific negatively charged residues in galectin-3's IDR are crucial for sensing pH changes and modulating condensation.
• These residues may also prevent the IDR from forming rapid, extensive aggregates.

Conclusions:

• Cation-π, π-π, and electrostatic interactions orchestrate protein condensation between disordered and structured domains.
• Sparse negatively charged residues in prion-like IDRs play a vital role in regulating pH-dependent phase separation.