Plasminogen activator inhibitor type 2 : a unique serpin with two mobile loops

Abstract

The superfamily of serine protease inhibitors (serpins) is a large group of proteins with diverse functions but a common tertiary structure. Active serpins are highly metastable molecules. Metastability is the property underlying the success and ubiquitousness of serpins. However, serpin metastability also accounts for improper conformational changes in serpin mutants which may result in pathological serpin polymerization. Plasminogen activator inhibitor type 2 (PAI-2) is a member of the subfamily of ov-serpins. It is the only wild-type (wt) serpin that spontaneously polymerizes under physiological conditions. Another unique feature of PAI-2 is the loop connecting helices C and D (the CD-loop), which is the longest among known serpins and is involved in interactions with the environment. Two partially overlapping goals were achieved during this thesis. The first was to study the molecular determinants involved in PAI-2 polymerization. By using in vitro mutagenesis in combination with biochemical and fluorescence methods, we have shown that wt PAI-2 exists both in stable monomeric and in polymerogenic conformations. The polymerogenic conformation can spontaneously form loop-sheet polymers and does not require conformational rearrangements prior to polymerization. The polymerogenic conformation of PAI-2 has an open β-sheet A, and it is stabilized by a disulfide bond formed between the unique CD-loop of PAI-2 and the bottom of the molecule. Under reducing conditions, the polymerogenic conformation of PAI-2 converts to the stable monomeric form. The polymerogenic and the stable monomeric forms are fully interconvertible, depending on the redox status of the environment. The stable monomeric conformation can be stabilized by vitronectin disulfide-bound to the CD-loop of PAI-2. The most populated conformation of the stable monomeric form of PAI-2 is that with the CD-loop folded on the side of the molecule. However, a mall fraction of stable monomeric PAI-2 can also exist with the CD-loop oriented toward the bottom of the molecule. Thus, the CD-loop of PAI-2 is a mobile molecular switch that regulates PAI-2 conformation. The second goal of the thesis was to use PAI-2 as a model protein to develop methods for intramolecular distance measurements. An improved purification procedure, the stability of the protein and our understanding of its structure make PAI-2 an attractive candidate for use as a model protein. In this context, we have used PAI-2 successfully to measure distances by the previously used DDEM and newly developed PDDEM methods