Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability

Abstract

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.

Conformational preludes to the latency transition in PAI-1 as determined by atomistic computer simulations and hydrogen/deuterium-exchange mass spectrometry

“…The details of how and where these unfolding events are initiated and how they are mechanistically linked to the process of latency transition in PAI-1 remains to be elucidated. In this respect it is interesting to note that limited proteolysis 20 and HDXMS experiments 21–23 have previously indicated high conformational flexibility in the area around helices D, E and F, which is often referred to as the “flexible joints” region (Fig. 1a).…”

“…Previous HDXMS experiments on PAI-1 were conducted at 25 °C or 37 °C 19, 21–23 . At these temperatures a number of peptides from the flexible joints region reach close to full deuteration within the first labeling time point.…”

Resolving distinct molecular origins for copper effects on PAI-1

“…Several factors influence the rate at which PAI-1 converts to the latent form, including ligands [ 24 , 25 ], post-translational modifications [ 26 ], pH [ 27 ], and protein dynamics [ 28 , 29 ]. Protein dynamics play a role in the latency process, whereby conditions that stabilize PAI-1 decrease protein dynamics, and those that destabilize PAI-1 increase protein dynamics [ 29 ]. Furthermore, hydrogen–deuterium exchange mass spectrometry (HDX-MS) has revealed local unfolding events within the hydrophobic core of PAI-1 that are hypothesized to be on the path to the latent state [ 30 ].…”

Copper in the suprachiasmatic circadian clock: A possible link between multiple circadian oscillators

“…Changes in cellular redox state in turn can influence membrane excitability, such as through modulating K + currents. Cu has been shown to interact with extracellular and intracellular signal transduction mechanisms, including modulating NMDA, MAPK, and TrkB signaling, a variety of ion channels, and extracellular matrix/proteases (e.g., collagen lyase, PAI‐1), all of which again can alter membrane excitability (Bucci et al., , ; D'Ambrosi & Rossi, ; Fujie et al., ; Migocka, ; Scheiber et al., ; Urso & Maffia, ; Zlatic et al., ). Our data are consistent with this, showing Cu/Cu depletion‐induced effects involving NMDA, MAPK, TrkB, and NO.…”