Resolving distinct molecular origins for copper effects on PAI-1

Joel C Bucci,Kayla D Mcconnell,Cynthia B Peterson,Joseph P Emerson,Gregory L Ware,Carlee S Mcclintock,Yuzhuo Chu

doi:10.1007/s00775-017-1489-5

Abstract

Components of the fibrinolytic system are subjected to stringent control to maintain proper hemostasis. Central to this regulation is the serpin plasminogen activator inhibitor-1 (PAI-1), which is responsible for specific and rapid inhibition of fibrinolytic proteases. Active PAI-1 is inherently unstable and readily converts to a latent, inactive form. The binding of vitronectin and other ligands influences stability of active PAI-1. Our laboratory recently observed reciprocal effects on the stability of active PAI-1 in the presence of transition metals, such as copper, depending on the whether vitronectin was also present (Thompson et al. Protein Sci 20:353–365, 2011). To better understand the molecular basis for these copper effects on PAI-1, we have developed a gel-based copper sensitivity assay that can be used to assess the copper concentrations that accelerate the conversion of active PAI-1 to a latent form. The copper sensitivity of wild-type PAI-1 was compared with variants lacking N-terminal histidine residues hypothesized to be involved in copper binding. In these PAI-1 variants, we observed significant differences in copper sensitivity, and these data were corroborated by latency conversion kinetics and thermodynamics of copper binding by isothermal titration calorimetry. These studies identified a copper-binding site involving histidines at positions 2 and 3 that confers a remarkable stabilization of PAI-1 beyond what is observed with vitronectin alone. A second site, independent from the two histidines, binds metal and increases the rate of the latency conversion.

Highlights

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor responsible for controlling blood flow, and is subjected to strict regulation at the genetic as well as protein level [1]
While this result clearly establishes that a metal-binding site exists near the N terminus of PAI-1, the key question is to determine whether copper binding effects the transition between active and latent forms of PAI-1
Copper binds to the N-terminal histidines of PAI-1 (H2, H3), but binding to this site does not result in accelerated latency in the presence of copper

Summary

Introduction

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) responsible for controlling blood flow, and is subjected to strict regulation at the genetic as well as protein level [1]. PAI-1 inhibits tissue-type (tPA) and urokinase (uPA) plasminogen activators (PAs) [1]. The inhibitory mechanism of PAI-1 mirrors the process of peptide bond cleavage by serine proteases until the final step, in which the acylated RCL inserts into the protein body, translocating the protease with a highly distorted active site to the opposite pole of the inhibitor [14]. PAI-1 has a half-life of approximately 1–2 h, spontaneously undergoes a significant structural rearrangement to an inactive, latent form that can no longer inhibit proteases (Fig. 1) [16]. VN binds to PAI-1 with high affinity (Kd ~0.1 nM) to localize and stabilize PAI-1 in the active form [22, 23]

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