Mapping of a conformational epitope on plasminogen activator inhibitor-1 by random mutagenesis. Implications for serpin function.
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 approximately 100-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 beta-sheet A, and together, these data have significant implications for the general serpin mechanism of proteinase inhibition.
Gorlatova
Natalia V
NV
Department of Vascular Biology, The Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA.
Elokdah
Hassan
H
Fan
Kristi
K
Crandall
David L
DL
Lawrence
Daniel A
DA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018604:Epitope Mapping; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000276:immunology; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D015843:Serpins / Q000502:physiology*; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D015843:Serpins