Dissecting molecular details and functional effects of the high-affinity copper binding site in plasminogen activator Inhibitor-1
Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor for plasminogen activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). As a unique member in the serine protease inhibitor (serpin) family, PAI-1 is metastable and converts to an inactive, latent structure with a half-life of 1-2 hr under physiological conditions. Unusual effects of metals on the rate of the latency conversion are incompletely understood. Previous work has identified two residues near the N-terminus, H2 and H3, which reside in a high-affinity copper-binding site in PAI-1 [Bucci JC, McClintock CS, Chu Y, Ware GL, McConnell KD, Emerson JP, Peterson CB (2017) J Biol Inorg Chem 22:1123-1,135]. In this study, neighboring residues, H10, E81, and H364, were tested as possible sites that participate in Cu(II) coordination at the high-affinity site. Kinetic methods, gel sensitivity assays, and isothermal titration calorimetry (ITC) revealed that E81 and H364 have different roles in coordinating metal and mediating the stability of PAI-1. H364 provides a third histidine in the metal-coordination sphere with H2 and H3. In contrast, E81 does not appear to be required for metal ligation along with histidines; contacts made by the side-chain carboxylate upon metal binding are perturbed and, in turn, influence dynamic fluctuations within the region encompassing helices D, E, and F and the W86 loop that are important in the pathway for the PAI-1 latency conversion. This investigation underscores a prominent role of protein dynamics, noncovalent bonding networks and ligand binding in controlling the stability of the active form of PAI-1.
Publication Source (Journal or Book title)
Protein science : a publication of the Protein Society
Chu, Y., Bucci, J. C., & Peterson, C. B. (2021). Dissecting molecular details and functional effects of the high-affinity copper binding site in plasminogen activator Inhibitor-1. Protein science : a publication of the Protein Society, 30 (3), 597-612. https://doi.org/10.1002/pro.4017