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

• Published in: Fibrinolysis & proteolysis 1999-07, Vol.13 (4), p.203-207
• Main Authors: Willems, P.K.A., Gils, A., Aertgeerts, K., De Ranter, C.J., Declerck, P.J.
• Format: Article
• Language: English
• ISSN: 1369-0191; 1532-222X
• DOI: 10.1016/S0268-9499(99)90072-3

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.