Engineering a membrane-binding protein to trimerize and induce high membrane curvature

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Annexins are Ca2+-dependent proteins involved in membrane repair.
• Annexin A4 (ANXA4) and Annexin A5 (ANXA5) form trimers that induce membrane curvature, aiding repair.
• Annexin A3 (ANXA3), despite homology to ANXA4, does not form trimers.

Purpose of the Study:

• To engineer Annexin A3 (ANXA3) to form trimers and induce membrane curvature.
• To investigate the role of trimer formation and membrane curvature in annexin-mediated membrane repair.
• To identify factors beyond trimerization and curvature essential for effective membrane repair.

Main Methods:

• Molecular dynamics simulations to engineer ANXA3 mutations.
• Atomic force microscopy to analyze protein-membrane interactions.
• Laser-induced membrane poration in cells to assess repair function.

Main Results:

• Engineered ANXA3 mutant successfully formed trimers and induced high membrane curvature, similar to ANXA4.
• The engineered ANXA3 formed crystalline arrays on supported lipid membranes.
• Despite trimer formation and curvature induction, the engineered ANXA3 did not localize to membrane lesions and failed to repair them.

Conclusions:

• Trimer formation and negative membrane curvature induction are insufficient for annexin-mediated membrane repair.
• High membrane binding affinity is a critical, necessary factor for effective membrane repair by annexins.
• This study elucidates key determinants of annexin function in membrane repair.