RESUMO
Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN. The study presented here dissects the changes in molecular dynamics underlying the destabilizing effects of Cu(II) on PAI-1. We utilize backbone amide hydrogen/deuterium exchange monitored by mass spectrometry to assess PAI-1 dynamics in the presence and absence of Cu(II) ions with and without the SMB domain of VN. We show that Cu(II) produces an increase in dynamics in regions important for the function and overall stability of PAI-1, while the SMB domain elicits virtually the opposite effect. A mutant form of PAI-1 lacking two N-terminal histidine residues at positions 2 and 3 exhibits similar increases in dynamics upon Cu(II) binding compared to that of active wild-type PAI-1, indicating that the observed structural effects are not a result of coordination of Cu(II) to these histidine residues. Finally, addition of Cu(II) results in an acceleration of the local unfolding kinetics of PAI-1 presumed to be on pathway to the latency conversion. The effect of ligands on the dynamics of PAI-1 adds another intriguing dimension to the mechanisms for regulation of PAI-1 stability and function.
Assuntos
Cobre/metabolismo , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Medição da Troca de Deutério/métodos , Fibrinólise , Histidina/química , Humanos , Cinética , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Somatomedinas/química , Somatomedinas/metabolismo , Resposta a Proteínas não Dobradas , Vitronectina/química , Vitronectina/metabolismoRESUMO
UNLABELLED: Plasminogen activator inhibitor-1 (PAI-1) is a biologically important serine protease inhibitor (serpin) that, when overexpressed, is associated with a high risk for cardiovascular disease and cancer metastasis. Several of its ligands, including vitronectin, tissue-type and urokinase-type plasminogen activator (tPA, uPA), affect the fate of PAI-1. Here, we measured changes in the solvent accessibility and dynamics of an important unresolved functional region, the reactive center loop (RCL), upon binding of these ligands. Binding of the catalytically inactive S195A variant of tPA to the RCL causes an increase in fluorescence, indicating greater solvent protection, at its C-terminus, while mobility along the loop remains relatively unchanged. In contrast, a fluorescence increase and large decrease in mobility at the N-terminal RCL is observed upon binding of S195A-uPA to PAI-1. At a site distant from the RCL, binding of vitronectin results in a modest decrease in fluorescence at its proximal end without restricting overall loop dynamics. These results provide the new evidence for ligand effects on RCL conformation and dynamics and differences in the Michaelis complex with plasminogen activators that can be used for the development of more specific inhibitors to PAI-1. This study is also the first to use electron paramagnetic resonance (EPR) spectroscopy to investigate PAI-1 dynamics. SIGNIFICANCE: Balanced blood homeostasis and controlled cell migration requires coordination between serine proteases, serpins, and cofactors. These ligands form noncovalent complexes, which influence the outcome of protease inhibition and associated physiological processes. This study reveals differences in binding via changes in solvent accessibility and dynamics within these complexes that can be exploited to develop more specific drugs in the treatment of diseases associated with unbalanced serpin activity.
Assuntos
Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vitronectina/metabolismo , Sítios de Ligação , Domínio Catalítico , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Vitronectina/químicaRESUMO
The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1), is an important biomarker for cardiovascular disease and many cancers. It is therefore a desirable target for pharmaceutical intervention. However, to date, no PAI-1 inhibitor has successfully reached clinical trial, indicating the necessity to learn more about the mechanics of the serpin. Although its kinetics of inhibition have been extensively studied, less is known about the latency transition of PAI-1, in which the solvent-exposed reactive center loop (RCL) inserts into its central ß-sheet, rendering the inhibitor inactive. This spontaneous transition is concomitant with a large translocation of the RCL, but no change in covalent structure. Here, we conjugated the fluorescent probe, NBD, to single positions along the RCL (P13-P5') to detect changes in solvent exposure that occur during the latency transition. The results support a mousetrap-like RCL-insertion that occurs with a half-life of 1-2 h in accordance with previous reports. Importantly, this study exposes unique transitions during latency that occur with a half-life of â¼5 and 25 min at the P5' and P8 RCL positions, respectively. We hypothesize that the process detected at P5' represents s1C detachment, while that at P8 results from a steric barrier to RCL insertion. Together, these findings provide new insights by characterizing multiple steps in the latency transition.