Importance of the hinge region between alpha-helix F and the main part of serpins, based upon identification of the epitope of plasminogen activator inhibitor type 1 neutralizing antibodies

The serpin plasminogen activator inhibitor type 1 (PAI-1) is an important p rotein in the regulation of fibrinolysis and inhibits its target proteinase s through formation of a covalent complex. In the present study, we have id entified the epitope of two PAI-1 neutralizing monoclonal antibodies (MA-33 H1F7 and MA-55F4C12). Based upon differential cross-reactivity data of thes e monoclonals with PAI-1 from different species and on a sequence alignment between these PAI-1s, combined with the three-dimensional structure, we pr edicted that the residues Glu(128)-Val(129)-Glu(130)-Arg(131) and Lys(154) (at the hinge region between alpha-helix F and the main part of the PAI-1-m olecule) might form the major site of interaction. Therefore a variety of a lanine mutants were generated and evaluated for their affinity toward both monoclonal antibodies. The affinity constants of MA-55F4C12 and MA-33HIF7 f or PAI-1 were 2.1 +/- 1.6 x 10(9) M-1 and 5.4 +/- 1.7 x 10(9) M-1, respecti vely, but decreased between 13- and 270-fold upon mutation of Lys(154) to A la(154) or Glu(128)-Val(129)-Glu(130)-Arg(131) to Ala-Ala-Ala-Ala. The comb ined mutations (PAI-1-EVER/K), however, resulted in an absence of binding t o either of the antibodies. Both antibodies bound to PAI-1-wt/t-PA complexe s with a similar affinity as to PAI-1-wt (K-A = 4-5 x 10(9) M-1). The epito pe localization reveals the molecular basis for the neutralizing properties of both monoclonal antibodies. In addition, it provides new insights into the validity of various models that have been proposed for the serpin/prote inase complex, excluding full insertion of the reactive-site loop.