Structural Basis for Recognition of Urokinase-type Plasminogen Activator by Plasminogen Activator Inhibitor-1

Abstract

Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissuetype PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1⅐uPA(S195A) Michaelis complex and present its crystal structure at 2.3-Å resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4 -P3 residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 ␤-sheet B, and the 147-loop directly contacts PAI-1 ␤-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies.

Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1

“…In addition, the relative orientation between tPA and PAI-1 is quite different from its uPA homologue (Fig. 3) (15).…”

“…Our structure shows that this residue does not interact with the 37-loop and may have a weak van der Waals contact (4.2 Å to Pro-E149) with the 147-loop of tPA-SPD. For comparison, Glu-I351 is critical in the uPA⅐PAI-1 Michaelis complex formation (15), forming a strong (2.7Å) hydrogen bond with the side chain phenolic group of Tyr-E149.…”

“…The structural origin of PAI-1 specificity to uPA was revealed by the crystal structure of the uPA⅐PAI-1 Michaelis complex that we determined in 2011 (15). Compared with uPA, tPA is more susceptible to PAI-1 inhibition with a faster second order rate constant (2.6 ϫ 10 7 M Ϫ1 s Ϫ1 versus 4.8 ϫ 10 6 M Ϫ1 s Ϫ1 ) (16).…”

Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

“…Inhibition of the fibrinolytic system in vivo may occur either at the level of plasmin by ␣ 2 -antiplasmin or ␣ 2 -macroglobulin. Inhibition can also be at the level of tPA/ uPA (3,4), mainly by plasminogen activator inhibitor type-1 (PAI-1), which forms a suicide complex with tPA or uPA (5).…”

“…The tertiary structure of PAI-1 is composed of nine ␣-helices (hA-hI) and three ␤-sheets (A, B, and C). The key feature is the reactive center loop (RCL), a solvent-exposed unstructured loop of ϳ20 amino acids containing the target sequence recognized by proteases such as tPA and uPA (5)(6)(7)(8).…”