MOLECULAR EVOLUTION OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 FUNCTIONAL STABILITY

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine pr otease inhibitor (serpin) supergene family and a central regulatory pr otein in the blood coagulation system. PAI-1 is unique among serpins i n exhibiting distinct active and inactive (latent) conformations in vi vo. Though the structure of latent PAI-I was recently solved, the stru cture of the short-lived, active form of PAI-1 is not known, In order to probe the structural basis for this unique conformational change, a randomly mutated recombinant PAI-1 expression library was constructed in bacteriophage and screened for increased functional stability, Fou rteen unique clones were selected, and shown to exhibit functional hal f-lives (T(1/2)s) exceeding that of wild-type PAI-1 by up to 72-fold. The most stable variant (T-1/2 = 145 h) contained four mutations. Deta iled analysis of these four mutations, individually and in combination , demonstrated that the markedly enhanced functional stability of the parent compound mutant required contributions from all four substituti ons, with no individual T-1/2 exceeding 6.6 h, The functional stabilit y of at least eight of the remaining 13 compound mutants also required interactions between two or more amino acid substitutions, with no si ngle variant increasing the T-1/2 by >10-foId, The nature of the ident ified mutations implies that the unique instability of the PAI-1 activ e conformation evolved through global changes in protein packing and s uggest a selective advantage for transient inhibitor function.