Contribution of conserved lysine residues in the alpha2-antiplasmin C terminus to plasmin binding and inhibition

Abstract

α(2)-Antiplasmin is the physiological inhibitor of plasmin and is unique in the serpin family due to N- and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn-410 to Lys-464, and the lysine residues (Lys-418, Lys-427, Lys-434, Lys-441, Lys-448, and Lys-464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C terminus of α(2)-antiplasmin, we systematically and sequentially mutated the C-terminal lysines, studied the effects on the rate of plasmin inhibition, and measured the binding affinity for plasmin via surface plasmon resonance. We determined that the C-terminal lysine (Lys-464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C terminus of α(2)-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between α(2)-antiplasmin and plasmin. When the C terminus of α(2)-antiplasmin was removed, the binding affinity for active site-blocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin.

FIGURE 1.

Schematic representation of the C terminus of α₂-antiplasmin and positions at which mutations were introduced. Full-length WT α₂-antiplasmin and various mutants (Lys-to-Ala mutations or truncation) of the C terminus were generated, expressed, and purified. Dashes represent the wild-type sequence, and the introduction of a stop codon is indicated (Δ).

FIGURE 2.

SI of plasmin by recombinant human α₂-antiplasmin. Plasmin (1 nm) was incubated with WT or mutant α₂-antiplasmin (0.2–1.5 nm) for 1 h at 37 °C. Residual protease activity was measured in the presence of H-Ala-Phe-Lys-AMC (0.2 mm). A, WT recombinant α₂-antiplasmin; B, P414stop (CtermΔ) mutant α₂-antiplasmin; C, K464A mutant α₂-antiplasmin; D, K434A/K441A/K448A/K464A mutant α₂-antiplasmin; E, mean SI of plasmin by WT and mutant recombinant α₂-antiplasmin.

FIGURE 3.

Plasmin inhibition by recombinant human α₂-antiplasmin. A, progress curves of plasmin inhibition using plasmin (0.5 nm) and WT α₂-antiplasmin (1–2.5 nm) in the presence of H-Ala-Phe-Lys-AMC (1 mm). Nonlinear regression analysis using Equation 1 was used to determine the first-order rate constant (k_obs). B, the k_obs is plotted against the WT α₂-antiplasmin concentration, and linear regression analysis was performed to determine the uncorrected second-order rate constant (k′). To account for substrate inhibition, Equation 2 was applied to determine the corrected second-order rate constant (k_a). C, progress curves of plasmin inhibition using plasmin (0.5 nm) and P414stop (CtermΔ) α₂-antiplasmin (80–140 nm) in the presence of H-Ala-Phe-Lys-AMC (1 mm). the k_obs was obtained as described above. D, the k_obs is plotted against the CtermΔ α₂-antiplasmin concentration to determine the k′. The k_a of CtermΔ α₂-antiplasmin was calculated using Equation 2.

FIGURE 4.

Effect of single Lys-to-Ala mutations in the human α₂-antiplasmin C terminus on plasmin inhibition. A, mean rate of plasmin inhibition (k_a) by WT and mutant recombinant α₂-antiplasmin (n = 3). B, mean k_a of plasmin for WT and mutant recombinant α₂-antiplasmin.

FIGURE 5.

Effect of sequential Lys-to-Ala mutations and truncations in the human α₂-antiplasmin C terminus on plasmin inhibition. A, mean rate of plasmin inhibition (k_a) by WT and mutant recombinant α₂-antiplasmin (n = 3). B, mean k_a of plasmin for WT and mutant recombinant α₂-antiplasmin.

FIGURE 6.

Sensorgrams of the binding of recombinant human α₂-antiplasmin to active site-blocked plasmin measured by surface plasmon resonance. WT or mutant recombinant α₂-antiplasmin (20 nm) was immobilized on a NTA chip. The binding of various concentrations of active site-blocked plasmin to α₂-antiplasmin was monitored in real time. A, binding of active site-blocked plasmin (2–8 nm) to WT α₂-antiplasmin (χ² = 0.21). B, binding of active site-blocked plasmin (20–120 nm) to P414stop (CtermΔ) recombinant α₂-antiplasmin (χ² = 0.60). C, binding of active site-blocked plasmin (4–20 nm) to K464A mutant α₂-antiplasmin (χ² = 0.31). D, binding of active site-blocked plasmin (20–100 nm) to K434A/K441A/K448A/K464A mutant α₂-antiplasmin (χ² = 0.54).

FIGURE 7.

Effect of mutations in the α₂-antiplasmin C terminus on binding to active site-blocked plasmin as studied via surface plasmon resonance. A, mean binding affinity (K_D) of WT and mutant recombinant α₂-antiplasmin for active site-blocked plasmin (n = 3). B, mean K_D and association and dissociation constants of WT and mutant recombinant α₂-antiplasmin for active site-blocked plasmin.

FIGURE 8.

Mechanism of serpin inhibition. I represents serpin, and E represents the protease. The forward rate constant of serpin with protease is represented as k₁. The association rate constant (k_a1) measured in surface plasmon resonance studies is equal to k₁. k₋₁ is the reverse rate constant. The rate at which conformational change (EI*) occurs is denoted as k₂. k₃ occurs during the substrate reaction, which results in cleaved serpin and release of active protease (E + I*). Formation of the covalent protease·serpin complex (E-I*) results in complete inhibition, and the rate constant is represented as k₄.