Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• The serpin superfamily includes key protease inhibitors.
• A conserved feature of inhibitory serpins is a small, uncharged residue at the P14 position.
• Plasminogen activator inhibitor type 1 (PAI-1) is a crucial regulator of fibrinolysis.

Purpose of the Study:

• To investigate the role of the P14 residue in PAI-1's inhibitory function.
• To determine how mutations at P14 affect PAI-1's interaction with proteases.
• To elucidate the structural requirements for PAI-1's conversion from inhibitor to substrate.

Main Methods:

• Site-directed mutagenesis of PAI-1 at the P14 position.
• Assays for inhibitory activity against tissue-type and urokinase-type plasminogen activators.
• Analysis of protease-inhibitor complex formation and cleavage sites.
• Thermal denaturation studies to assess protein stability.
• Functional analysis of recombinant PAI-1 variants with diverse P14 substitutions.

Main Results:

• Mutating P14 Thr-333 to Arg abolished PAI-1's inhibitory activity.
• Mutant PAI-1 bound proteases similarly to wild-type but was cleaved as a substrate.
• Cleavage occurred at the P1-P1' bond in the mutant, unlike SDS-stable complex formation in wild-type.
• Charged residues at P14, regardless of size, converted PAI-1 into a substrate.
• Protein stability was similar in native forms but increased upon cleavage by elastase.

Conclusions:

• Protease binding is not sufficient for PAI-1 inhibition; reactive center loop insertion is critical.
• Charged residues at P14 hinder loop insertion, leading to substrate conversion.
• The P14 residue plays a vital role in regulating PAI-1's inhibitory mechanism.
• Understanding P14's role provides insights into serpin regulation and potential therapeutic targets.