Crystal Structure of Plasminogen Activator Inhibitor-1 in an Active Conformation with Normal Thermodynamic Stability

Abstract

The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure. Here we report the crystal structure of PAI-1 W175F at 2.3 Å resolution as the first model of the metastable native molecule. Structural comparison with a quadruple mutant (14-1B) previously used as representative of the active state uncovered key differences. The most striking differences occur near the region that houses three of the four mutations in the 14-1B PAI-1 structure. Prominent changes are localized within a loop connecting ␤-strand 3A with the F helix, in which a previously observed 3 10 -helix is absent in the new structure. Notably these structural changes are found near the binding site for the cofactor vitronectin. Because vitronectin is the only known physiological regulator of PAI-1 that slows down the latency conversion, the structure of this region is important. Furthermore, the previously identified chloridebinding site close to the F-helix is absent from the present structure and likely to be artifactual, because of its dependence on the 14-1B mutations. Instead we found a different chlorine-binding site that is likely to be present in wild type PAI-1 and that more satisfactorily accounts for the chlorine stabilizing effect on PAI-1.

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

“…For that reason, most crystal structures of PAI-1 are of latent PAI-1 or of stabilized mutants (9, 18 -20). However, recently, the crystal structure of W175F PAI-1 has been determined, which may be the structure most closely representing active PAI-1 (6).…”

“…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).…”

Molecular mechanism of two nanobodies that inhibit PAI‐1 activity reveals a modulation at distinct stages of the PAI‐1/plasminogen activator interaction

“…As expected, a major part of the RCL, residues 335 to 348, is disordered in the electron density maps and is therefore not included in the final model of the complex. Comparison of the PAI‐1‐W175F structure in the complex and the isolated PAI‐1‐W175F structure (PDB: 3Q02, chain A) shows that no major conformational changes are induced upon interaction of PAI‐1 with the inhibitory Nbs (Cα RMSD of 0.678 Å).…”

“…The obtained diffraction data were processed using autoPROC in default settings, with a high‐resolution cutoff on CC 1/2 0.60 . The data were initially phased by molecular replacement using the structure of PAI‐1‐W175F (Protein Data Bank [PDB]: 3Q02) or PAI‐1‐stab (PDB: 1DB2) in active conformation as a search model using PHASER . Nb42 and Nb64 were initially modelled based on homologous Nb structures that were selected using a protein basic local alignment search tool (BLAST) (PDB: 5JA8 and 5JA9, respectively).…”

Remarkable extension of PAI-1 half-life surprisingly brings no changes to its structure

“…Another recent PDB entry 3pb1 describes the complex of the PAI-1 4 and EWA SKRZYPCZAK-JANKUN 14-1B mutant with the enzymatically inactive uPA S195A mutant, giving valuable information about the complex formation between these molecules (6). The most recently deposited structure PDB ID: 3q02 contains only one mutation (W198F) and has stability superior over these listed above (t 1/2 ~400 h and 18 times better than wt-PAI-1 at the same conditions) (7) but like 1oc0 does not depict the reactive loop due to a disorder (missing |355-370|) (8). Natural PAI-1 is glycosylated at two asparagines: 232 and 288, but except for cleaved PAI-1 where it was confirmed in the crystal structure (PDB ID: 1a7c, with pentapeptides mimicking A4-β-strand thus having a molecule core as in the latent form) (9), this information is otherwise not provided.…”