Plasminogen activator inhibitor 1 (PAI-1) in its active conformation: crystallization and preliminary X-ray diffraction data.

Because of its intrinsic lability, wild-type plasminogen activator inhibitor 1 (PAI-1) cannot be crystallized in its active conformation. Therefore, a stable variant of PAI-1 was used to retain the active conformation during crystallization. Four different crystallization conditions were evaluated in detail and two major types of crystals were detected. Whereas solutions consisting of either (i) cacodylate and sodium acetate, (ii) lithium sulfate and polyethylene glycol 4K, or (iii) imidazole, sodium chloride and sodium potassium phosphate buffer revealed thin platelet crystals, a solution (iv) containing ammonium acetate, citrate and polyethylene glycol 4K appeared to enhance the formation of clustered brush-like crystals. Crystals grown under condition (iii) were found to be suitable for X-ray data collection and consequent structural investigation. Data collection was 79.8% complete with a maximum resolution of 2.92 A. Importantly, PAI-1 retained its functional properties under all conditions.

Identification of positively charged residues contributing to the stability of plasminogen activator inhibitor 1.

Plasminogen activator inhibitor 1 (PAI-1), a member of the serpins, has a unique conformational flexibility. A typical characteristic is its intrinsic lability resulting in the conversion of the active conformation to a latent conformation. In the present study, we have evaluated the effect of substitution of positively charged residues located at the turn connecting strand s4C with strand s3C, either with negatively charged or with neutral residues, on the functional stability of PAI-1. The following mutants were constructed, purified and characterized in comparison to wild-type (wt) PAI-1: PAI-1-R186E,R187E (Arg186--> Glu and Arg187--> Glu), PAI-1-H190E,K191E (His190--> Glu and Lys191--> Glu) and PAI-1-H190L,K191L (His190--> Leu and Lys191--> Leu). In contrast to wtPAI-1 the mutants exhibited no inhibitory activity. Whereas latent wtPAI-1 can be reactivated (up to a specific activity of 78+/-19%) by treatment with guanidinium chloride, a similar treatment applied to these mutants resulted in a significant but relatively small increase of specific activity (i.e. to 14%). Evaluation of the functional stability (at 37 degrees C, pH 5.5, 1 M NaCl revealed a strongly decreased functional stability compared to wtPAI-1 (i.e. 3-9 h for the mutants vs. > 24 h for wtPAI-1). Further characterization by heat denaturation studies and plasmin susceptibility confirmed that removal or reversal of the positive charge on the turn connecting s4C with s3C results in PAI-1 mutants with a highly accelerated conversion of active to latent forms. We can therefore conclude that the pronounced positive charge in the turn connecting s4C with s3C is of the highest importance for the functional stability of PAI-1.

Rational design of complex formation between plasminogen activator inhibitor-1 and its target proteinases.

Considerable progress in understanding the mechanism of inhibition of proteinases by serpins has been obtained from different biochemical studies. These studies reveal that stable serpin/proteinase complex formation involves insertion of the reactive-site loop of the serpin and occurs at the acyl-enzyme stage. Even though no three-dimensional structure of a serpin/proteinase complex is resolved, structural information is available on some of the individual compounds. Molecular modeling techniques combined with recently acquired biochemical/biophysical data were used to provide insight into the stable complex formation between plasminogen activator inhibitor-1 (PAI-1) and the target proteinases: tissue-type plasminogen activator, urokinase-type plasminogen activator, and thrombin. This study reveals that PAI-1 initially interacts with its target proteinase when its reactive-site loop is solvent exposed and thereby accessible for the proteinase. Stable complex formation, however, involves the insertion of the reactive-site loop up to P7 and results in a tight binding geometry between PAI-1 and its target proteinase. The influence of different biologically relevant molecules on PAI-1/proteinase complex formation and the differences in inhibition rate constants observed for the different proteinases can be explained from these models.

Mechanisms contributing to the conformational and functional flexibility of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is unique among the serine proteinase inhibitors (serpins) in that it can adopt at least three different conformations (active, substrate and latent). We report the X-ray structure of a cleaved substrate variant of human PAI-1, which has a new beta-strand s4A formed by insertion of the amino-terminal portion of the reactive-site loop into beta-sheet A subsequent to cleavage. This is in contrast to the previous suggestion that the non-inhibitory function of substrate-type serpins is mainly due to an inability of the reactive-site loop to adopt this conformation. Comparison with the structure of latent PAI-1 provides insights into the molecular determinants responsible for the transition of the stressed active conformation to the thermostable latent conformation.

Crystallization and X-ray diffraction data of the cleaved form of plasminogen activator inhibitor-1.

To characterize the structural requirements for the conformational flexibility in plasminogen activator inhibitor-1 (PAI-1) we have crystallized human PAI-1, carrying a mutation which stabilizes PAI-1 in its substrate form. Crystallization was performed by the hanging drop diffusion method at pH 8.5 in the presence of 19% (w/v) polyethyleneglycol 4000 as a precipitant. The crystals appear after 3 days at 23 degrees C and belong to the monoclinic space group C2 with cell dimensions of a = 151.8 A, b = 47.5 A, c = 62.7 A, and beta = 113.9 degrees, and one molecule in the asymmetric unit. The X-ray diffraction data set contains data with a limiting resolution of 2.5 A. Biochemical analysis of the redissolved crystals indicated that during the crystallization process, cleavage had occurred in the active site loop at the P1-P1' position. The availability of good-quality crystals of the cleaved form of this serpin will allow its three-dimensional structure to be solved and will provide detailed information on the structure-function relationship in PAI-1.

A model of the reactive form of plasminogen activator inhibitor-1.

A model of the reactive form of plasminogen activator inhibitor-1 (PAI-1) has been constructed using molecular graphics and starting from the known crystal structure of latent PAI-1. The residues P16 to P10', of which P16-P4 form strand 4 of the beta-sheet A (s4A) and P3-P10' form an extended loop in the latent form, have been removed and remodeled into this structure, based on the structures of ovalbumin and cleaved alpha 1-proteinase inhibitor. Residues P4'-P10' were remodeled as a beta-strand s1C, located on the surface of the molecule and the N-terminal end (P16-P14) of the eliminated loop was rebuilt using appropriate backbone dihedrals. Subsequently, a secondary structure prediction program was applied and further optimization of the model was performed by several molecular dynamics runs. Apparently the beta-strand was stabilized by only two hydrogen bonds. Further analysis revealed that, although s4A was removed, s3A and s5A did not approach each other. In this current model it was also found that the large gap between the loop connecting s4C-s3C and the loop connecting s3B-hG remained 11 A in contrast to the small gap (4A) at a similar position in other serpins. These observations may explain the ease of a conformational change of the reactive site loop of PAI-1 during transition to the latent and the preinserted form. In addition the current model can be used for the design of stable, functional, PAI-1 mutants. Detailed structural analysis of the latter may facilitate studies on the structure-function relationship in PAI-1 in particular and in other serpins in general.