Latency and Substrate Binding Globally Reduce Solvent Accessibility of Plasminogen Activator Inhibitor Type 1 (PAI-1)

Abstract

Plasminogen activator inhibitor type 1 (PAI-1) plays key regulatory roles in fibrinolysis, cell migration, and tissue remodeling. A regulatory protein without known catalytic activity, PAI-1 modulates plasminogen activators through protein-protein interactions. Although global conformational alterations that occur in PAI-1 determine its regulatory activity, comprehensive assessments of concurrent dynamic, structural, and functional alterations of this critical regulatory protein have not yet been clearly defined. X-ray crystallographic studies have described four distinct PAI-1 conformational states: active, latent, reactive center loop peptide-annealed (RCL-PA), and cleaved mutant. In this study, backbone amide hydrogen-deuterium exchange detected by mass spectrometry was used to characterize dynamic and structural alterations of human PAI-1 (hPAI-1) in relation to its function. hPAI-1 conformers were defined by surface mapping the solvent-accessible sites for strategic secondary structural components of the protein. We observed a global protection from solvent for a majority of peptides in the latent conformer relative to the active conformer. Significant differences were observed in the RCL, helix A, helix D, and sheet 1C, and these regions were markedly more dynamic or solvent-exposed in the active conformation. The RCL-PA form adopts an intermediate conformational state between the active and the latent conformers. Our results demonstrate that the most dynamic regions of PAI-1 (the RCL, helices D and A, and sheet 5A) are flexible in the transition toward latency. They also show that the dynamic surface structures of the active, latent, and peptide-annealed conformers of PAI-1 are underestimated by theoretical solvent accessibility calculations derived from crystallographic data. Plasminogen activator inhibitor type 1 (PAI-1)1 is a key regulator of fibrinolysis, cell migration, and tissue remodeling (1-3). As a member of the serine protease inhibitor family, PAI-1 serves as a major inhibitor of the proteases tissue plasminogen activator and urokinase plasminogen activator (uPA) (4, 5). Recent clinical studies suggest important links between PAI-1 and accelerated development of atherosclerosis and a prothrombotic state, with attendant enhanced risks for development of myocardial infarction, stroke, and thromboembolic events (6 -9). Animal model studies further demonstrate that PAI-1 plays a critical role in ventricular remodeling following an acute myocardial infarction, a process important in the development of heart failure (10). The many links between PAI-1, cardiovascular disease, and thrombosis have thus made this regulatory protein a recent focus of intense research interest. Despite this, the structural and dynamic conformational alterations that underlie the regulatory functions of PAI-1 are incompletely understood.In vivo, synthesized PAI-1 is secreted and bound to an adhesive, stabilizing glycoprotein, vitronectin, in a noncovalent complex (11). PAI-1 bound to vitronectin remains folded in an "activ...

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local Flexibility of Plasminogen Activator Inhibitor 1 upon Binding to the Somatomedin B Domain of Vitronectin

“…The question of whether these differences are related to the different functional properties of the three serpins remains unknown. PAI-1 was also investigated previously using HDX, but inconsistencies in the reported data render a comparison with the results reported here problematic.…”

Single fluorescence probes along the reactive center loop reveal site-specific changes during the latency transition of PAI-1

“…Despite an array of studies that include extensive mutagenesis, thermodynamic characterization, crystallization, HDX-MS, and use of RCL-mimicking peptides and monoclonal antibodies, the timescale for events during the transition from active to latent conformation of PAI-1 is still not well defined. 1,2,6,18,20,[26][27][28][29][30][31][32][33][34][35][36][37][38][39] We thereby conjugated the fluorescent NBD reporter to single positions along the RCL and observed the changes that occur during this process. Significant effects on the stability or fold were not observed nor expected for these mutants (Fig.…”

Demonstration of Three Distinct High-Molecular-Weight Complexes between Plasminogen Activator Inhibitor Type 1 and Tissue-Type Plasminogen Activator

“…These sequences correspond to the exosite loops of hA, s1B-hB-hC, s3C-s4B-s5B, and s6A, 28 respectively, at the N-terminus. Additionally, in the upper band, the sequence of E 244 -K 263 , which corresponds to hG and hH, 28 was also absent, both of which were shown to move closer to urokinase type PA (uPA) upon complex formation with uPA. 29 At this time, no clear differences in the crystal structures among different types of high-molecular-weight complexes have been reported.…”

“…16 The PAI-1 sequences of V 1 -R 18 , N 31 -K 65 , F 208 -K 243 , and G 264 -K 277 were missing in both bands. These sequences correspond to the exosite loops of hA, s1B-hB-hC, s3C-s4B-s5B, and s6A, 28 respectively, at the N-terminus. Additionally, in the upper band, the sequence of E 244 -K 263 , which corresponds to hG and hH, 28 was also absent, both of which were shown to move closer to urokinase type PA (uPA) upon complex formation with uPA.…”