Abstract
The contribution of Arg(129) of the serpin, antithrombin, to the mechanism of allosteric activation of the protein by heparin was determined from the effect of mutating this residue to either His or Gln. R129H and R129Q antithrombins bound pentasaccharide and full-length heparins containing the antithrombin recognition sequence with similar large reductions in affinity ranging from 400- to 2500-fold relative to the control serpin, corresponding to a loss of 28-35% of the binding free energy. The salt dependence of pentasaccharide binding showed that the binding defect of the mutant serpin resulted from the loss of approximately 2 ionic interactions, suggesting that Arg(129) binds the pentasaccharide cooperatively with other residues. Rapid kinetic studies showed that the mutation minimally affected the initial low affinity binding of heparin to antithrombin, but greatly affected the subsequent conformational activation of the serpin leading to high affinity heparin binding, although not enough to disfavor activation. Consistent with these findings, the mutant antithrombin was normally activated by heparin for accelerated inhibition of factor Xa and thrombin. These results support an important role for Arg(129) in an induced-fit mechanism of heparin activation of antithrombin wherein conformational activation of the serpin positions Arg(129) and other residues for cooperative interactions with the heparin pentasaccharide so as to lock the serpin in the activated state.
Highlights
Antithrombin, a plasma glycoprotein belonging to the serpin superfamily of proteins, is a critical regulator of the proteolytic enzymes of blood coagulation, especially thrombin and factor Xa [1, 2]
The ability of heparin to accelerate antithrombin-proteinase reactions is dependent on the binding to the inhibitor of a unique pentasaccharide sequence in the polysaccharide chain (10 –12). Heparin chains containing this sequence bind the fully glycosylated ␣-form of antithrombin with high affinity (KD of 10 –20 nM at physiological ionic strength and pH) and induce an activating conformational change in the inhibitor. Whereas this conformational change is sufficient to activate antithrombin to rapidly inhibit factor Xa, the acceleration of antithrombin inhibition of thrombin requires in addition a longer heparin chain to bridge the inhibitor and proteinase in a ternary complex
We have elucidated the contribution of Arg129 of antithrombin to heparin binding and activation of the serpin
Summary
Antithrombin, a plasma glycoprotein belonging to the serpin (serine proteinase inhibitor) superfamily of proteins, is a critical regulator of the proteolytic enzymes of blood coagulation, especially thrombin and factor Xa [1, 2]. The ability of heparin to accelerate antithrombin-proteinase reactions is dependent on the binding to the inhibitor of a unique pentasaccharide sequence in the polysaccharide chain (10 –12) Heparin chains containing this sequence bind the fully glycosylated ␣-form of antithrombin with high affinity (KD of 10 –20 nM at physiological ionic strength and pH) and induce an activating conformational change in the inhibitor. The x-ray crystal structures of free and heparin-complexed antithrombin [13,14,15] together with molecular modeling and biochemical studies [16, 17] have suggested that heparin activates antithrombin by expelling the reactive bond loop from the center of -sheet A of the protein core in which it is partially buried in the native structure This results in the complete exposure of the loop in a manner similar to that found in other native serpin structures and presumably in a conformation optimal for proteinase interaction [15]. Neither Arg nor Lys125 was found to be essential for activation of the serpin, Arg did contribute to stabilizing the activated conformation [20, 21]
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