K. Aertgeerts

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°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.

Induction of conformational changes within crystals of plasminogen activator inhibitor-1 (PAI-1)

Abstract Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin (serine protease inhibitor) superfamily, that can adopt three different conformations: active, latent and substrate. It was recently shown that the non-ionic detergent Triton X-100 (TX-100) can accelerate the conformational transitions in wild-type PAI-1 in solution. Recently, we have crystallized a stable PAI-1 variant (PAI-1-stab) in its active conformation (Acta Cryst D 55, 574–576, 1999). In this study, we examined the effect of TX-100 on the conformational transitions in PAI-1-stab, in solution as well as in crystals. Within the crystal (at t = 0: 75 ± 3% active, 10 ± 4% non-reactive and 15 ± 4% substrate; mean ± SD, n = 3) a time-dependent increase of the substrate form was observed, with a concomitant decrease of the active form. A steady state situation with active (52 ± 2%) and substrate (34 ± 5%) forms was reached within 4 h. Under those conditions, the amount of non-reactive PAI-1 remained essentially unchanged (14 ± 5%). The conformational changes induced by TX-100 in PAI-1-stab in solution (at t = 0: 59 ± 2% active, 0% non-reactive and 41 ± 2% substrate, n = 3) were characterized mainly by a decrease of the active form in favour of the non-reactive form, reaching a steady state between 15 and 24 h resulting in active (20 ± 3%, n = 4), substrate (29 ± 3%) and non-reactive (48 ± 5%) forms. Thus, this study demonstrates that, even though characterized with a restricted mobility, conformational changes can be induced within protein crystals. Importantly, in both cases studied a steady state situation with various functional forms was observed.

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.