Metals affect the structure and activity of human plasminogen activator inhibitor-1. II. Binding affinity and conformational changes

✍ Scribed by Lawrence C. Thompson; Sumit Goswami; Cynthia B. Peterson

• Book ID: 105356582
• Publisher: Cold Spring Harbor Laboratory Press
• Year: 2011
• Tongue: English
• Weight: 311 KB
• Volume: 20
• Category: Article
• ISSN: 0961-8368
• DOI: 10.1002/pro.567

✦ Synopsis

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 μ__M__, 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.