Engineering of conformations of plasminogen activator inhibitor-1 - A crucial role of beta-strand 5A residues in the transition of active form to latent and substrate forms

The serpin (serine proteinase inhibitor) family is of general protein chemi cal interest because of its ability to undergo large conformational changes , in which the surface-exposed reactive centre loop (RCL) is inserted as st rand 4 in the large central beta-sheet A. Loop insertion is an integral par t of the inhibitory mechanism and also takes place at conversion of serpins to the latent state, occurring spontaneously only in plasminogen activator inhibitor-1 (PAI-1). We have investigated the importance of beta-strand 5A residues for the activity and latency transition of PAI-1. An approximatel y fourfold increase in the rate of latency transition resulted from His-sub stitution of Gln324 (position 334 in the alpha(1)-proteinase inhibitor temp late numbering), which interacts with the underlying alpha-helix B. The sid e chains of Gln321 and Lys325 (template residues 331 and 335, respectively) form hydrogen bonds to the peptide backbone of a loop connecting alpha-hel ix F and beta-strand 3A. While substitution with Ala of Glu321 had only min or effects on the properties of PAI-1, substitution with Ala of Lys325 led to stabilization of the inhibitory activity at incubation conditions leadin g to conversion of wild-type PAI-1 to a substrate form, and to an anomalous reaction towards a monoclonal antibody, which induced a delay in the laten cy transition of the mutant, but not wild-type PAI-1. We conclude that the anchoring of P-strand 5A plays a crucial role in loop insertion. These find ings provide new information about the mechanism of an important example of protein conformational changes.