W0030

Active Plasminogen Activator Inhibitor-1 Structure Reveals Differences from Latent and Cleaved Forms. Allan M. Sharp, Penelope E. Stein, Daniel A. Lawrence, Randy J. Read, University of Alberta, Department of Biochemistry and Department of Medical Microbiology and Immunology, 1-41 Med. Sci. Bldg., Edmonton, AB T6G 2H7, CANADA

The serpin plasminogen activator inhibitor-1 (PAI-1) functions biologically in three forms: active, latent, and cleaved. Like all functional members of the serpin (serine proteinase inhibitor) family, it inhibits a serine proteinase, plasminogen activator, undergoing a structural rearrangement in the process. The active forms of serpins contain a large surface loop, which is bound and cleaved by the target proteinase, presumably allowing the structure rearrangements to the cleaved form. The N-terminal fragment of the active site loop is inserted into the central beta-sheet of the protein, moving its end by ~50 Å. The extremely strong complex formed between serpins and their targets is presumed to be due to the distortion and trapping of the acyl-enzyme complex by this rearrangement. PAI-1 can insert its active site loop into the beta sheet without undergoing cleavage, leading to the latent form. This slow process plays a role in its biological regulation. As the principal regulator of plasminogen activator, PAI-1 plays an important role in vascular fibrinolysis, and many other functions involving tissue remodelling, including angiogenesis and inflammation.

Prior studies have revealed the structures of latent and cleaved PAI-1^1,2. This solution of the structure of a quadruple mutant of PAI-1, deficient in the latency transition, marks the first time the active, latent, and cleaved forms of a single serpin can be compared. At 2.97 Å resolution, the conformation of the active site loop can be determined, and other features of the structure can be observed. This allows comparisons to be made among the three structures with regard to mechanism, and the binding of cofactor molecules such as vitronectin. The active site loop is compared to other known loops, and the possible effects of the mutations causing retardation of the latency transition are also investigated. Several predictions of PAI-1's active conformation based on solution studies are compared with the structure, and theories about why PAI-1 is the only natural latent-state forming serpin are evaluated.

¹Mottonen, J. et al. Nature 355, 270-273 (1992).

² Aertgeerts, K, et al. Nature Structural Biology 2, 891, 897(1995).