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 tissue-type 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-1·uPA(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 β-sheet B, and the 147-loop directly contacts PAI-1 β-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.
Highlights
The urokinase-type plasminogen activator2 system is composed of uPA, its cognate receptor, and two spe
The ability of plasminogen activator inhibitor (PAI)-1 to and quickly recognize its target protease has long been a subject of extensive research
We have presented the structure of the noncovalent Michaelis complex between Plasminogen activator inhibitor-1 (PAI-1) and uPA and revealed the detailed mechanism at the atomic level
Summary
Protein Expression and Purification—The catalytically inactive form of the human uPA protease domain (Ile-16 –Glu244 in chymotrypsin numbering) containing the S195A mutation was expressed in Pichia pastoris yeast strain X-33 and purified as described previously [27]. The PAI-11⁄7uPA complex was subsequently eluted with buffer A containing 300 mM imidazole and dialyzed against 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The complex was further purified using a Superdex-75 column (GE Healthcare). The data were processed and scaled using the HKL2000 suite [31] These crystals have hexagonal shape and belong to the P3221 space group with one complex in the crystallographic asymmetric unit. Phasing and Refinement—The structure of the PAI-11⁄7uPA complex was solved by molecular replacement using MolRep [32] with structures of the active stable variant of PAI-1 (Protein Data Bank code 1DVM) [17] and the uPA catalytic domain (code 2O8T) as searching models.
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