Structures of Active and Latent PAI-1:  A Possible Stabilizing Role for Chloride Ions

Abstract

Serpins exhibit a range of physiological roles and can contribute to certain disease states dependent on their various conformations. Understanding the mechanisms of the large-scale conformational reorganizations of serpins may lead to a better understanding of their roles in various cardiovascular diseases. We have studied the serpin, plasminogen activator inhibitor 1 (PAI-1), in both the active and the latent state and found that anionic halide ions may play a role in the active-to-latent structural transition. Crystallographic analysis of a stable mutant form of active PAI-1 identified an anion-binding site between the central beta-sheet and a small surface domain. A chloride ion was modeled in this site, and its identity was confirmed by soaking crystals in a bromide-containing solution and calculating a crystallographic difference map. The anion thus located forms a 4-fold ligated linchpin that tethers the surface domain to the central beta-sheet into which the reactive center loop must insert during the active-to-latent transition. Timecourse experiments measuring active PAI-1 stability in the presence of various halide ions showed a clear trend for stabilization of the active form with F(-) > Cl(-) > Br(-) >> I(-). We propose that the "stickiness" of this pin (i.e., the electronegativity of the anion) contributes to the energetics of the active-to-latent transition in the PAI-1 serpin.

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

“…The binding of vitronectin to PAI-1 decreases the rate at which PAI-1 converts to this latent form. Stable forms of PAI-1 that do not collapse to the latent form at an appreciable rate have been engineered by incorporating amino acid replacements at a variety of loci, including the core ␤-sheet (16,27,38,54,55). The stable variant of PAI-1 that has been best characterized and has yielded the crystallographic structure of active PAI-1 (55) is the 14-1B mutant with the mutations N152H, K156T, Q321L, and M356I (16,38).…”

“…Stable forms of PAI-1 that do not collapse to the latent form at an appreciable rate have been engineered by incorporating amino acid replacements at a variety of loci, including the core ␤-sheet (16,27,38,54,55). The stable variant of PAI-1 that has been best characterized and has yielded the crystallographic structure of active PAI-1 (55) is the 14-1B mutant with the mutations N152H, K156T, Q321L, and M356I (16,38). Although most functions are indistinguishable for wild-type and the stable 14-1B PAI-1, recent work suggests that there are subtle differences in structure and function (35,56).…”

“…However, these contrary experiments used different reagents than the ones we have employed in this study. Notably, the other experiments used denatured vitronectin, along with the stable mutant of PAI-1 (14-1B) (16,38), which is often used by experimentalists and deemed to be equivalent to wild-type PAI-1. As seen in Figs.…”

“…PAI-1 shares the common structural features and mechanism of inhibition characteristic of the other inhibitory members of the serpin family (15,16). The tertiary structure of PAI-1 is composed of nine ␣-helices (hA-hI), three ␤-sheets (A, B, and C), and a solvent-exposed unstructured loop of ϳ20 amino acids referred to as the reactive center loop, because it carries the inhibitory reactive peptide bond.…”

“…The tertiary structure of PAI-1 is composed of nine ␣-helices (hA-hI), three ␤-sheets (A, B, and C), and a solvent-exposed unstructured loop of ϳ20 amino acids referred to as the reactive center loop, because it carries the inhibitory reactive peptide bond. The overall scaffold of the molecule is defined by the five-stranded central ␤-sheet A. PAI-1 spontaneously alters in conformation, converting from the active state to an energetically more favorable inactive latent state (16,17,19), where the reactive center loop translocates into the central ␤-sheet.…”