Engineering thermostability in serine protease inhibitors

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Native serine protease inhibitors (serpins) exhibit inherent strain due to unfavorable structural interactions.
• Human alpha(1)-antitrypsin, a model serpin, displays strain from side-chain overpacking, buried polar groups, and cavities.
• Stabilizing single amino acid substitutions in alpha(1)-antitrypsin alleviate strain by removing these unfavorable interactions.

Purpose of the Study:

• To determine if engineering rules derived from alpha(1)-antitrypsin apply to other clinically significant serpins.
• To enhance the conformational stability of other serpin family members.

Main Methods:

• Introduced amino acid substitutions in human alpha(1)-antichymotrypsin and human antithrombin III at positions analogous to stabilizing sites in alpha(1)-antitrypsin.
• Assessed thermostability changes following these substitutions.
• Utilized mutational analysis and structural examination to understand the impact of substitutions.

Main Results:

• Two-thirds of the tested amino acid substitutions successfully increased thermostability across all examined serpins.
• Mutational and structural analyses indicated common suboptimal folding strategies across different serpin members.
• While overall strategies are shared, specific structural details can vary between individual serpins.

Conclusions:

• Serpins share common, albeit suboptimal, folding strategies, providing a basis for engineering stability.
• Engineering principles identified in alpha(1)-antitrypsin are transferable and effective for improving the stability of other serpins.
• These findings offer a valuable framework for the rational design of more stable serpin therapeutics.