Biophysical characterization of anticoagulant hemextin AB complex from the venom of snake Hemachatus haemachatus

Abstract

Hemextin AB complex from the venom of Hemachatus haemachatus is the first known natural anticoagulant that specifically inhibits the enzymatic activity of blood coagulation factor VIIa in the absence of factor Xa. It is also the only known heterotetrameric complex of two three-finger toxins. Individually only hemextin A has mild anticoagulant activity, whereas hemextin B is inactive. However, hemextin B synergistically enhances the anticoagulant activity of hemextin A and their complex exhibits potent anticoagulant activity. In this study we characterized the nature of molecular interactions leading to the complex formation. Circular dichroism studies indicate the stabilization of beta-sheet in the complex. Hemextin AB complex has an increased apparent molecular diameter in both gas and liquid phase techniques. The complex formation is enthalpically favorable and entropically unfavorable with a negative change in the heat capacity. Thus, the anticoagulant complex shows less structural flexibility than individual subunits. Both electrostatic and hydrophobic interactions are important for the complexation; the former driving the process and the latter helping in the stabilization of the tetramer. The tetramer dissociates into dimers and monomers with the increase in the ionic strength of the solution and also with increase in the glycerol concentration in the buffer. The two dimers formed under each of these conditions display distinct differences in their apparent molecular diameters and anticoagulant properties. Based on these results, we have proposed a model for this unique anticoagulant complex.

FIGURE 1

Conformational changes associated with the formation of hemextin complex. CD spectra of (A) hemextin A (0.5 mM) and (B) hemextin B (0.5 mM). (C) Conformational changes in hemextin A with increasing concentrations of hemextin B: (black) hemextin A 0.5 mM; (red) hemextin A 0.5 mM plus hemextin B 0.25 mM; (green) hemextin A 0.5 mM plus hemextin B 0.4 mM; (blue) hemextin A 0.5 mM plus hemextin B 0.5 mM; (gray) hemextin A 0.5 mM plus hemextin B 0.9 mM; (inset) the observed change in the CD spectra around the 217-nm region.

FIGURE 2

Measurement of molecular diameter during the hemextin AB complex formation using GEMMA. The molecular diameters of the individual hemextins and the hemextin AB complex are calculated based on their electrophoretic mobility. The formation of hemextin AB complex leads to an increase in the molecular diameter. Addition of equimolar toxin C does not show any significant increase in the molecular diameters of hemextin A and hemextin B validating the obtained data.

FIGURE 3

Determination of hydrodynamic diameter using DLS. (A) CONTIN analysis hemextin A, hemextin B, and hemextin AB complex in 50 mM Tris-HCl buffer. Effect of various concentrations of NaCl (B) and glycerol (C) on hemextin AB complex. The calculated hydrodynamic diameters for each molecular species are shown. (Note the dimer formed in glycerol has a different diameter than the one formed in salt.)

FIGURE 4

Interaction studies between hemextin A and B using ITC. (A) Raw ITC data showing heat release upon injections of 1 M hemextin B into a 1.4-ml cell containing 0.1 mM of hemextin A; (B) integration of the raw ITC data yields the heat/mol versus molar ratio. The best values of the fitting parameters are 1.04 for N, 2.23 × 10⁶ M⁻¹ for K_A, and −11.68 kcal M⁻¹ for ΔH (Table 1).

FIGURE 5

Thermodynamics of hemextin A-hemextin B interaction. (A) Effect of temperature on the energetics of hemextin A-hemextin B interaction: enthalpy change (ΔH), change in entropy term (TΔS), and free energy change (ΔG). (B) Enthalpy-entropy compensation in complex formation. (Point of intersection of lines corresponding to ΔH and ΔG corresponds to T_s)

FIGURE 6

Hemextin AB complex formation under different buffer conditions. (A) Dependence of K_A on the ionic strength of the buffer. The binding affinity decreases with the increase in buffer ionic strength indicating the importance of electrostatic interactions. (B) Dependence of K_A on the glycerol concentration. The binding affinity decreases with the increase in glycerol concentration indicating the importance of hydrophobic interactions.

FIGURE 7

SEC studies of hemextin AB complex in different buffer conditions. (A) Elution profiles of hemextin AB complex in Tris-HCl buffer. Effect of various concentrations of NaCl (B) and glycerol (C) on hemextin AB complex. The tetrameric complex (peak denoted by 4) dissociates into dimer and monomer (peaks denoted by 2 and 1, respectively) with the increase in salt or glycerol. Asterisk denotes the peak containing conformationally altered hemextin A. (D) Calibration of the column using the following proteins as molecular weight markers: ovomucoid (28 kDa), ribonuclease (15.6 kDa), cytochrome C (12 kDa), apoprotinin (7 kDa), and pelovaterin (4 kDa). The molecular weights of the tetramer, dimer, and monomers were calculated from the calibration curve.

FIGURE 8

Effect of buffer conditions on anticoagulant activity. Effect of various concentrations of NaCl (A) and glycerol (B) on the anticoagulant activity of hemextin A (O), hemextin B (M), hemextin AB complex (▴). The anticoagulant activity of hemextin AB complex decreases with the increase in concentrations of NaCl and glycerol. The arrows indicate the concentrations of (A) NaCl and (B) glycerol where the anticoagulant complex exists mostly as a mixture of dimer and monomers (see text for details); (▪) denotes the anticoagulant activity recorded in the absence of the proteins.

FIGURE 9

One-dimensional ¹H NMR studies. Spectrum of (A) hemextin A and hemextin B under different buffer conditions. Note that in the presence of salt, hemextin A undergoes a conformational change; also, the peaks are sharp throughout the spectrum. In addition, all 1D ¹H NMR spectra also exhibited a wide range of chemical shift dispersions (amide region 7–10 ppm, Hα region 3.8–6 ppm, and methyl region −0.4 –to 1.5 ppm), which is a characteristic of well-folded proteins. Therefore, the observed structural change is not due to resonance broadening or aggregation (because no aggregation state was observed as mentioned) but it is only due to addition of NaCl to hemextin A.

FIGURE 10

A proposed model of hemextin AB complex. (A) Schematic diagram depicting the formation of hemextin AB complex. Hemextins A and B, two structurally similar three-finger toxins, form a compact and rigid tetrameric complex with 1:1 stoichiometry (24). (B) Schematic diagram showing the effect of salt and glycerol on conformations of hemextins A and B. Hemextin A undergoes a conformational change in the presence of salt. (C) Dissociation of the tetrameric hemextin AB complex in the presence of salt and glycerol. The dissociation probably occurs in two different planes. Thus the hemextin AB dimer in high salt is different from the dimer formed in the presence of glycerol. Two putative anticoagulant sites are shown with dotted semicircles (see text for details).