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 lM, 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.

Zn2+ Mediates High Affinity Binding of Heparin to the αC Domain of Fibrinogen

“…4). The increase in RU values is attributed to conformational changes in fibrinogen that occur upon Zn 2ϩ binding that alter the refractive index of fibrinogen (24,25). Based on analysis of the plot of R eq values versus Zn 2ϩ concentration, Zn 2ϩ binds fibrinogen with a K d value of 9.4 Ϯ 2.2 M, a value similar to the K d of 18 M reported previously (12).…”

Single fluorescence probes along the reactive center loop reveal site-specific changes during the latency transition of PAI-1

“…Whether hF/s3A loop displacement/repositioning or passage of the RCL through the gate region is the rate‐limiting step of the latency process is not apparent from this study and will require further investigation. The results, which leverage site‐specific probes at P5′ and P8, reveal intermediate events in the latency transition that provide important details relevant to our previous approach‐to‐equilibrium kinetic study on metal binding to PAI‐1 that demonstrate one or more conformational intermediates that occur after metals bind and induce the latency transition . Furthermore, they expand on data from recent molecular modeling using variable path sampling that support a long‐lived prelatent intermediate in the active‐to‐latent transition from molecular modeling using variable path sampling .…”

“…The results, which leverage site-specific probes at P5 0 and P8, reveal intermediate events in the latency transition that provide important details relevant to our previous approach-to-equilibrium kinetic study on metal binding to PAI-1 that demonstrate one or more conformational intermediates that occur after metals bind and induce the latency transition. 8,48 Furthermore, they expand on data from recent molecular modeling using variable path sampling that support a longlived prelatent intermediate in the active-to-latent transition from molecular modeling using variable path sampling. 49 Consistent with our findings, the molecular modeling study documents detachment of s1C as a requisite for the prelatent intermediate and a high-energy barrier involving displacement of hF that must be overcome to reach the final latent state.…”

Crystal Structure of Plasminogen Activator Inhibitor-1 in an Active Conformation with Normal Thermodynamic Stability

“…It should be noted that adding submicromolar concentrations of either divalent zinc or copper ions to the crystallization drops caused immediate precipitation, supporting a specific interaction of the metals with PAI-1. This specific metal binding that leads to a conformational change may be relevant to recently published work that shows a destabilization of the active conformation of PAI-1 by copper, nickel, or cobalt and metal effects that are modulated by vitronectin binding to PAI-1 (32,33).…”