The Contribution of the Exosite Residues of Plasminogen Activator Inhibitor-1 to Proteinase Inhibition

Abstract

The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and ␤-trypsin by PAI-1. Alanine substitutions at the distal P4 (Glu-350) and P5 (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k a ) and overall inhibition (k app ) with tPA by 13.4-and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k lim ) increased by 3.3-fold. The effects of double mutations on k a , k lim , and k app were small with uPA and nonexistent with ␤-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into ␤-sheet A. Moreover, mutational analysis indicates that the P5 residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.Plasminogen activator inhibitor-1 (PAI-1) 1 functions as the primary regulator of the fibrinolytic system by inhibiting the conversion of plasminogen into plasmin via its action on tissuetype plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (1). This inhibitor also plays a vital role in other physiological processes, including tumor invasion and tissue remodeling (2). PAI-1 belongs to the serpin superfamily, which shares several unique structural features, including a five-stranded ␤-sheet motif and a flexible reactive center loop (RCL). The conformational changes associated with these structures have been linked to the inhibitory function of PAI-1 (3-9). Notably, the mechanism of inhibition depends on the structural changes accompanying the S (stressed) to R (relaxed) transition that results in complete insertion of the Nterminal (proximal) part of the RCL into ␤-sheet A as an additional ␤-strand, s4A. The proteinase, tethered by a covalent bond with the P1 residue of the serpin, is displaced from the initial docking site to the opposite end of the serpin molecule, separating the P1Ј and P1 residues by ϳ70 Å (10 -12). This mechanism efficiently distorts and inactivates the catalytic triad of the proteinase, stabilizing the serpin-proteinase complex at the acyl-enzyme intermediate stage.Unique to the PAI-1 inhibitory mechanism are the exosite i...

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

“…The residue Glu-I351 of PAI-1 was previously shown by mutational studies to implicate in the tPA binding (43), although the opposite conclusion was obtained in a different study (45). 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.…”

“…These three salt bridges are partially shielded away from solvent by the long 37-loop, potentially leading to lower dielectric constant in this region and thus enhancing these charged interactions. The importance of PAI-1 Glu-I350 in tPA binding was also previously identified by site-directed mutagenesis (43,44). The third region of PAI-1 interacting with tPA 37-loop is the P2Ј Ala-I348 of PAI-1, which forms a hydrogen bond with the side chain amino group of tPA Arg-E39.…”

Modulation of Serpin Reaction through Stabilization of Transient Intermediate by Ligands Bound to α-Helix F

“…To address this question, we have evaluated the effects of recombinant SMBD on the kinetics of RCL insertion and stoichiometry of the reaction of uPA and tPA with PAI-1 or its complex with anti-␣HF mAb MA-55F4C12, and compared the results with the data obtained previously for the whole Vn isolated from human plasma (45). According to the Scheme 1, bifurcation between inhibitory and substrate pathways is controlled by LDA; however, disruption of the pattern of the exosite interactions at the PAI-1/tPA interface, which changes significantly k lim for the reaction of PAI-1 with tPA, does not affect SI (31,50,51). Therefore, a step of the serpin mechanism, which is likely different from stabilization/ destabilization of LDA, contributes to the outcome of PAI-1 reaction.…”

“…Human recombinant PAI-1 and its S338C (P9 Cys) mutant variant were purified, labeled with an NBD group, and characterized as described elsewhere (54). NBD P9 E350A/E351A PAI-1, which was obtained and characterized as described elsewhere (50), was provided by Dr. C. Ibarra. The activity of PAI-1 was determined by titrating the FIGURE 1.…”

“…Disturbing the exosite interactions through either mutations at the RCL of PAI-1 (31,50) or through a decrease in pH (51) does not significantly affect the stoichiometry of inhibition for the reaction between PAI-1 and tPA. To determine whether or not exosite interactions between PAI-1 and proteinase contribute to changes in the SI for binary (PAI-1⅐mAb) and ternary (PAI-1⅐mAb⅐SMBD) complexes, a mutant variant of PAI-1 with glutamate to alanine mutation in positions P4Ј and P5Ј (E350A/ E351A) was studied.…”