Mapping of a Conformational Epitope on Plasminogen Activator Inhibitor-1 by Random Mutagenesis

Abstract

The mechanism for the conversion of plasminogen activator inhibitor-1 (PAI-1) from the active to the latent conformation is not well understood. Recently, a monoclonal antibody, 33B8, was described that rapidly converts PAI-1 to the latent conformation (Verhamme, I., Kvassman, J. O., Day, D., Debrock, S., Vleugels, N., Declerck, P. J., and Shore, J. D. (1999) J. Biol. Chem. 274, 17511-17517). In an attempt to understand this interaction, and more broadly to understand the mechanism of the natural transition of PAI-1 to the latent conformation, we have used random mutagenesis to identify the 33B8 epitope in PAI-1. This site involves at least 8 amino acids scattered over more than two-thirds of the linear sequence that form a compact epitope on the PAI-1 three-dimensional structure. Surface plasmon resonance studies indicate a high affinity interaction between latent PAI-1 and 33B8 that is ϳ100-fold higher than comparable binding to active PAI-1. Structural modeling results together with surface plasmon resonance analysis of parental and site-directed PAI-1 mutants with disrupted 33B8 binding suggest the existence of a specific PAI-1 intermediate structure that is stabilized by 33B8 binding. These analyses strongly suggest that this intermediate form of PAI-1 has a partial insertion of the reactive center loop into ␤-sheet A, and together, these data have significant implications for the general serpin mechanism of proteinase inhibition.

Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

“…Subsequent mutagenesis studies indicated that the epitope for MA-33B8 includes the loops between ␣-hD and ␤-s2A and between ␤-s2C and ␤-s3C and also includes the residues proceeding ␤-s3A (10,56). Additional characterization of this antibody using SPR experiments indicated about 100-fold greater affinity of MA-33B8 for latent PAI-1 compared with active PAI-1, a finding that was substantiated by an analysis of the active and latent x-ray structures indicating that the epitope is more compact in latent compared with active PAI-1 (56). Note the similarity to AZ3976, which, according to ITC and SPR measurements, binds latent PAI-1 with high affinity (K D 0.19 -0.29 M), whereas the affinity to active PAI-1 was negligible.…”

“…challenge for drug design; an opportunity because there are many conformational states of PAI-1 to which a molecule may interfere to achieve desired inhibition; a challenge because there are experimental difficulties to measure binding of an inhibitor to an isolated relevant PAI-1 form and to understand the mode of action and structure activity relationship. A simplified mechanistic scheme for PAI-1 latency conversion, partially adapted from earlier literature (20,56), is shown in Fig. 10.…”

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

“…PAI-1 mutagenesis and expression was performed as previously described (29). Cell extract was prepared from bacterial pellets by washing in ice cold 0.85% NaCl followed by centrifugation at 6,000 × g at 4°C (20 min).…”

“…The supernatant was collected as cell extract (CE). The concentrations of active mutant PAI-1 in CE were titrated as described (29), and the total protein concentration was measured using the BCA protein assay kit (Pierce Biotechnology). PAI-1 mutant inactivation by CDE-096 in CE was performed as described previously (33), with the total protein concentrations normalized using CE from nontransformed bacteria.…”

“…Under physiological conditions, the latency transition is irreversible; however, denaturant-induced refolding can reconstitute PAI-1 in the native, active form (26). Several latency-inducing agents have been described, including amphipathic nonionic detergents (27,28) and monoclonal antibodies or peptides (29)(30)(31). These latter agents are thought to stabilize a prelatent intermediate and thereby shift the structural equilibrium toward the latent conformation.…”