title: Metals affect the structure and activity of human plasminogen activator inhibitor-1. II. Binding affinity and conformational changes.
abstract: Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. The lifespan of the active form of PAI-1 is modulated via interaction with the plasma protein, vitronectin, and various metal ions. These metal ions fall into two categories: Type I metals, including calcium, magnesium, and manganese, stabilize PAI-1 in the absence of vitronectin, whereas Type II metals, including cobalt, copper, and nickel, destabilize PAI-1 in the absence of vitronectin, but stabilize PAI-1 in its presence. To provide a mechanistic basis for understanding the unusual modulation of PAI-1 structure and activity, the binding characteristics and conformational effects of these two types of metals were further evaluated. Steady-state binding measurements using surface plasmon resonance indicated that both active and latent PAI-1 exhibit a dissociation constant in the low micromolar range for binding to immobilized nickel. Stopped-flow measurements of approach-to-equilibrium changes in intrinsic protein fluorescence indicated that the Type I and Type II metals bind in different modes that induce distinct conformational effects on PAI-1. Changes in the observed rate constants with varying concentrations of metal allowed accurate determination of binding affinities for cobalt, nickel, and copper, yielding dissociation constants of ∼40, 30, and 0.09 μM, respectively. Competition experiments that tested effects on PAI-1 stability were consistent with these measurements of affinity and indicate that copper binds tightly to PAI-1.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, USA.
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Thompson
authors.forename: Sumit
authors.initials: S
authors.lastname: Goswami
authors.forename: Cynthia B
authors.initials: CB
authors.lastname: Peterson
mesh_terms: D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019216:Metals, Heavy / Q000737:chemistry* / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013696:Temperature
chemical_list: D002712:Chlorides; D019216:Metals, Heavy; D017395:Plasminogen Activator Inhibitor 1; C051321:SERPINE1 protein, human