Role of Arginine 132 and Lysine 133 in Heparin Binding to and Activation of Antithrombin
Journal of Biological Chemistry
The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Acceleration of the rate of inhibition of factor Xa involves a conformational change in antithrombin that is translated from the heparin binding site to the reactive center loop. A mechanism has been proposed for generation and propagation of the conformational change in which the binding of the negatively charged heparin reduces ionic repulsions between positively
... charged residues on and adjacent to the D-helix in the heparin binding site of antithrombin (van Boeckel, C. A. A., Grootenhuis, P. D. J., and Visser, A. (1994) Nature Struct. Biol. 1, 423-425). This charge neutralization is proposed to elongate the D-helix and initiate the conformational change which is then translated to the reactive center loop. Several basic residues, including arginine 132 and lysine 133, were predicted to be important both in heparin binding and in this mechanism of heparin activation. To test both the helix extension mechanism and the role of these two residues in heparin binding and factor Xa inhibition, we individually changed arginine 132 and lysine 133 to uncharged methionine by site-directed mutagenesis. The K d values for binding of R132M and K133M variants to the high affinity pentasaccharide were weakened only 2.3-and 4.5-fold respectively, suggesting a location for R132 and K133 peripheral to the main pentasaccharide binding site. However, the K d values for long chain high affinity heparin were weakened at least 17-fold for both R132M and K133M, indicating involvement of each residue in binding extended chain heparin species. These reductions in affinity were ionic strength-dependent. The rates of inhibition of factor Xa and thrombin by each variant, however, were indistinguishable from those of control antithrombin, and the accelerations of the rate of inhibition produced by heparin were normal. We conclude that neither arginine 132 nor lysine 133 plays an important role in the binding of heparin pentasaccharide or in the mechanism of heparin activation, suggesting that D-helix extension through charge neutralization is not the mechanism for transmission of conformational change from the heparin binding site to the reactive center region. Arginine 132 and lysine 133 do, however, play a role in tight binding of longer chain heparin species through ionic interactions.