title: Latency and substrate binding globally reduce solvent accessibility of plasminogen activator inhibitor type 1 (PAI-1). An adaptation of PAI-1 conformer crystal structures by hydrogen-deuterium exchange.
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.
authors.affiliation: Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
authors.forename: Benedicta N
authors.initials: BN
authors.lastname: Nukuna
authors.forename: Marc S
authors.initials: MS
authors.lastname: Penn
authors.forename: Vernon E
authors.initials: VE
authors.lastname: Anderson
authors.forename: Stanley L
authors.initials: SL
authors.lastname: Hazen
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D012997:Solvents