A spectroscopic study of the structures of latent, active and reactive-center-cleaved type-1 plasminogen-activator inhibitor.

Type-1 plasminogen-activator inhibitor (PAI-1) was studied by Fourier-transform infrared spectroscopy, far-ultraviolet CD spectroscopy, and fluorescence-emission spectroscopy, with the aim to obtain structural information about its active form. The spectra of latent, active and reactive-center-cleaved forms of PAI-1 produced by HT-1080 cells were different. While the cleaved and the latent forms were similar with regard to their beta-structure content, comparison of the spectra of these forms with the spectra of active PAI-1 suggested a much higher degree of unordered structure for the active form compared with the latent and reactive-center-cleaved forms than previously assumed. We discuss our results with reference to the known three-dimensional X-ray structures of latent PAI-1, of reactive-center-cleaved serpins, including reactive-center-cleaved PAI-1, and of intact serpins, and with reference to previous results on the differences in the affinity of mAbs for the different PAI-1 forms. We interpret our results in favor of a global rearrangement of secondary structure during latency transition and reactive-center cleavage in PAI-1, not only involving the reactive-center loop and parts of beta-sheets A and C, but also the "rear' side of the molecule, such as helices H and G. Thus, we suggest flexibility in serpin structural elements that were previously regarded as rigid.