Effects on human plasminogen conformation and activation rate caused by interaction with VEK-30, a peptide derived from the group A streptococcal M-like protein (PAM)

Abstract

In vertebrates, fibrinolysis is primarily carried out by the serine protease plasmin (Pm), which is derived from activation of the zymogen precursor, plasminogen (Pg). One of the most distinctive features of Pg/Pm is the presence of five homologous kringle (K) domains. These structural elements possess conserved Lys-binding sites (LBS) that facilitate interactions with substrates, activators, inhibitors and receptors. In human Pg (hPg), K2 displays weak Lys affinity, however the LBS of this domain has been implicated in an atypical interaction with the N-terminal region of a bacterial surface protein known as PAM (Pg-binding group A streptococcal M-like protein). A direct correlation has been established between invasiveness of group A streptococci and their ability to bind Pg. It has been previously demonstrated that a 30-residue internal peptide (VEK-30) from the N-terminal region of PAM competitively inhibits binding of the full-length parent protein to Pg. We have attempted to determine the effects of this ligand-protein interaction on the regulation of Pg zymogen activation and conformation. Our results show minimal effects on the sedimentation velocity coefficients (S degrees (20,w)) of Pg when associated to VEK-30 and a direct relationship between the concentration of VEK-30 or PAM and the activation rate of Pg. These results are in contrast with the major conformational changes elicited by small-molecule activators of Pg, and point towards a novel mechanism of Pg activation that may underlie group A streptococcal (GAS) virulence.

Figure 1

Effects of VEK-30 on Pm generation by uPA in Cl⁻- and acetate-containing buffers. Glu¹-Pg (27 nM) was activated by 20 nM of uPA in the presence of VEK-30. (A) Plots of absorbance at 405 nm versus time depicting the activity of generated Pm on the chromogenic substrate S-2251 (250 μM). Assays were performed in 10 mM HEPES/100 mM NaCl buffer, pH 7.4 (solid lines) or 10 mM HEPES/NaOAc buffer, pH 7.4 (dotted lines). For each buffer condition, the concentrations of VEK-30 included in the assays were, from bottom to top, 0, 0.9, 3, 15, and 75 μM. Inset, linearity of the rate of Pg activation at time points corresponding to ≤ 15% substrate consumption. Closed and open circles represent the data points obtained from assays conducted in NaCl and NaOAc buffers, respectively, while the lines correspond to the fits of those data obtained from linear regression analysis. (B) Fold increases in the rate of VEK-30 stimulated Pm generation in the presence of 10 mM HEPES/100 mM NaCl buffer, pH 7.4 (black bars) and 10 mM HEPES/100 mM NaOAc buffer, pH 7.4 (gray bars) as assessed from the initial rate data of companion panel A. Initial rates accompanying uPA-mediated Pm activation in the absence of VEK-30 in both Cl⁻- and acetate-containing buffers were used as the reference for the calculation of fold changes. Inset, concentration-response curves corresponding to the initial rates of Pm generation as a function of VEK-30 concentration. Data were fit by non-linear regression to the equation for one-site direct binding. The EC₅₀ values (± SE for the best fit) for VEK-30 derived from the depicted fits are 16.5 ± 0.9 μM and 1.8 ± 0.4 μM for the experiments conducted in Cl⁻ (black squares) and acetate (gray squares), respectively.

Figure 2

Effects of VEK-30 and sVEK-30 on the activation of Glu¹-Pg by SK in Cl⁻- and acetate-containing buffers. (A) Plots of absorbance at 405 nm versus time depicting the activity of generated Pm on the chromogenic substrate S-2251 (250 μM). Activation of Glu¹-Pg (27 nM) by the addition of SK (20 nM) was carried out in 10 mM HEPES/100 mM NaCl, pH 7.4 (solid lines), or 10 mM HEPES/100mM NaOAc buffer, pH 7.4 (dotted lines). For assays conducted in Cl⁻-containing buffer, the concentrations of VEK-30 used were, from bottom to top, 0, 0.3, 1.5, 15, 45, and 75 μM. For assays conducted in acetate-containing buffer, the traces depicted correspond to VEK-30 concentrations of 0, 1.5, 15, and 150 μM (bottom to top). Inset, linearity of the rate of Pg activation at time points corresponding to ≤ 15% substrate consumption. Closed squares represent the data points obtained from assays conducted in NaCl buffer, while the lines correspond to the fits of those data obtained from linear regression analysis. (B) The time course of Pm generation catalyzed by SK in the presence of sVEK-30 in Cl⁻- and acetate-containing buffers. Experimental conditions were the same as those described in Figure 2A. Assays conducted in Cl⁻-containing buffer are represented by the bottom grouping of traces, while those experiments conducted in acetate-containing buffer are denoted by the top three traces. For each buffer condition the concentrations of sVEK-30 used were 0 μM (solid lines), 15 μM (dashed lines), and 150 μM (dotted lines). (C) Fold increases in the rate of VEK-30-stimulated Pm generation in the presence of 10 mM HEPES/100 mM NaCl buffer, pH 7.4 (gray bars) and 10 mM HEPES/100 mM NaOAc buffer, pH 7.4 (white bars) as assessed from the initial rate data of companion panel A. Initial rates accompanying SK-mediated Pm activation in the absence of VEK-30 in both Cl⁻- and acetate-containing buffers were used as the reference for the calculation of fold changes. Inset, concentration-response curve corresponding to the initial rates of Pm generation as a function of VEK-30 concentration in Cl⁻-containing buffer. Data were fit by non-linear regression to the equation for one-site direct binding. The EC₅₀ value (± SE for the best fit) for VEK-30 derived from the depicted fit is 78.8 ± 11.6 μM.

Figure 3

The effects of VEK-30 on the rate of S-2251 conversion in the presence of catalytically active components of the Pg-SK system in Cl⁻-containing buffer. (A) Increasing amounts of VEK-30 enhance the rate of S-2251 (250 μM) hydrolysis in the presence of catalytic amounts of pre-formed SK-Pm complex (1.5 nM) and Glu¹-Pg (50 nM). The VEK-30 concentrations represented, from bottom to top, are 0, 15, 100, and 250 μM. Inset, Plot of the concentration-response data corresponding to the initial rates of S-2251 hydrolysis as a function of VEK-30. Data were fit by non-linear regression to the equation for one-site direct binding. The EC₅₀ value (± SE for the best-fit) for VEK-30 derived from these fitted data is 50.4 ± 1.3 μM. (B) Hydrolytic activity of 45 nM preformed SK-Pm complex (black traces) and 60 nM Pm (gray traces) on S-2251 (250 μM) in the presence of 0 μM (solid lines), 15 μM (dashed lines), and 150 μM (dotted lines) VEK-30. All experiments were conducted in 10 mM HEPES/100 mM NaCl, pH 7.4.

Figure 4

The interaction of Pg with wt-PAM and R¹⁰¹A/H¹⁰²A-PAM. (A) Effects of recombinant wt-PAM protein (solid lines) and R¹⁰¹A/H¹⁰²A-PAM (dotted lines) on the activation rate of Pg (80 nM) by equimolar amounts of SK. The concentrations of wt-PAM used were 0, 1, 5, 10 and 20 nM (bottom to top) and the concentrations of R¹⁰¹A/H¹⁰²A-PAM used were 1, 5, 10, 20 nM (bottom to top). The buffer was 10 mM HEPES/100 mM NaCl, pH 7.4. (B) Real time interaction (gray traces) of wt-PAM (0.1, 6.25, 12.5 and 35 nM, bottom to top) and immobilized Pg studied by SPR. The black traces are representative fits of the association and dissociation phases shown for the 6.25 nM concentration of wt-PAM. (C) Real time interaction (gray traces) of R¹⁰¹A /H¹⁰²A-PAM (0.1, 6.25, 12.5 and 35 nM, bottom to top) and immobilized Pg studied by SPR. The black traces are representative fits of the association and dissociation phases shown for the 6.25 nM concentration of the mutant version of PAM. For the SPR studies, Pg was immobilized on CM-5 chips (~600 RU) and the interactions with the PAM proteins were monitored at 25 °C using a flow rate of 30 μL/min in HBS-EP buffer.

Figure 5

Sedimentation equilibrium data showing the tendency of PAM (22 μM) to self-associate. The data (○) for this representative scan were collected at 20 °C at a rotor speed of 15,000 rpm. The calculated fit (black line) for these data yielded a M_w,app of 71600. The partial specific volume of PAM was calculated on the basis of amino acid composition and determined to be 0.734 mL/g. A simulation of the fit corresponding to monomeric PAM (M_w,app = 43600) under identical conditions is shown for comparison (gray line).