Loop-sheet mechanism of serpin polymerization tested by reactive center loop mutations

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Folding

Background:

• Serpins inhibit proteases via reactive center loop (RCL) insertion into beta-sheet A.
• Serpin folding bypasses a stable loop-inserted state, creating a metastable conformation.
• Misfolded serpins form polymers in the endoplasmic reticulum, linked to point mutations.

Purpose of the Study:

• To investigate the validity of the 20-year-old loop-sheet hypothesis of serpin polymerization.
• To characterize a variant of alpha-1-antitrypsin (AAT) with specific mutations in the RCL (P8-P6).
• To determine if these mutations affect polymerization propensity under various conditions.

Main Methods:

• Created an alpha-1-antitrypsin variant with aspartic acid substitutions at P8-P6 of the RCL.
• Tested polymerization of the variant upon incubation with peptides or RCL cleavage.
• Induced polymerization using guanidine HCl or heat.
• Assessed polymerization of Z and Siiyama AAT variants with Asp mutations in COS-7 cells.

Main Results:

• The P8-P6 Asp variant failed to polymerize with peptides or upon RCL cleavage.
• The variant readily polymerized under guanidine HCl or heat denaturation, similar to controls.
• Asp mutations did not impede polymerization of Z or Siiyama AAT variants in cell culture.

Conclusions:

• Results strongly refute the loop-sheet hypothesis as the sole mechanism of serpin polymerization.
• The P8-P6 region is likely a minor contributor to an extensive domain swap in serpin polymers.
• Serpin polymerization involves a more complex mechanism than previously proposed.