A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.

The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring ␤-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Bjö rk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-Å long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central ␤-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.

The dissociation equilibrium constant for heparin binding to antithrombin III (ATIII) is a measure of the cofactor's binding to and activation of the proteinase inhibitor, and its salt dependence indicates that ionic and non-ionic interactions contribute ϳ40 and ϳ60% of the binding free energy, respectively. We now report that phenylalanines 121 and 122 (Phe-121 and Phe-122) together contribute 43% of the total binding free energy and 77% of the energy of non-ionic binding interactions. The large contribution of these hydrophobic residues to the binding energy is mediated not by direct interactions with heparin, but indirectly, through contacts between their phenyl rings and the non-polar stems of positively charged heparin binding residues, whose terminal amino and guanidinium groups are thereby organized to form extensive and specific ionic and non-ionic contacts with the pentasaccharide. Investigation of the kinetics of heparin binding demonstrated that Phe-122 is critical for promoting a normal rate of conformational change and stabilizing AT*H, the high affinity-activated binary complex. Kinetic and structural considerations suggest that Phe-122 and Lys-114 act cooperatively through non-ionic interactions to promote P-helix formation and ATIII binding to the pentasaccharide. In summary, although hydrophobic residues Phe-122 and Phe-121 make minimal contact with the pentasaccharide, they play a critical role in heparin binding and activation of antithrombin by coordinating the P-helixmediated conformational change and organizing an extensive network of ionic and non-ionic interactions between positively charged heparin binding site residues and the cofactor.The sulfated polysaccharide heparin functions as an anticoagulant by binding to antithrombin III (ATIII) 1 and greatly accelerating its rates of thrombin and factor Xa inhibition.Heparin binding to ATIII is a two-step process consisting of an initial weak interaction that induces a protein conformational change leading to the formation of a high affinity binary complex with the cofactor (AT*H) and ATIII activation (1, 2). Functional investigations of chemically modified, naturally occurring mutant and recombinant antithrombins have identified Arg-47, Lys-114, Lys-125, and Arg-129 as the most important positively charged amino acid residues in the heparin binding site of . Direct interactions of these basic residues with negatively charged groups of heparin are observed in the crystal structure of an ATIII-pentasaccharide complex (13). Studies of heparin binding kinetics indicate that Lys-125 is the most important amino acid in the initial docking with heparin and that Arg-129, Lys-114, and Arg-47 are critical for the protein conformational change step leading to the high affinity, activated AT*H complex. Heparin binding leads to elongation of ATIII helix D by 1.5 turns at its carboxyl-terminal end as well as the formation of a new alpha helix, the P-helix, at its amino-terminal end (13,14). These structural changes are associated with expulsion of the P14...

The mechanism for heparin activation of antithrombin III has been postulated to involve disruption of interactions between its reactive loop P1 residue and Glu 255 on the underlying protein surface. To test this hypothesis, the potential P1-constraining Arg 393 -Glu 255 hydrogen bond and ionic interactions were eliminated by converting Glu 255 to alanine. E255A and wild-type ATIIIs have identical reactive loop sequences (including the P1 and P14 residues), but differ in that Glu 255 -mediated, P1-constraining interactions with the underlying surface cannot form in the mutant. Relative to its wildtype parent, E255A had a 5-fold higher affinity for heparin and pentasaccharide. In the absence of cofactor, E255A exhibited a 5-fold activation of thrombin inhibition but no activation of factor Xa inhibition. Pentasaccharide addition elicited no further activation of thrombin inhibition but increased the factor Xa inhibition rate 100-fold. E255A heparin-dependent thrombin and factor Xa inhibition rates were 1000-and 2-fold faster, respectively, than pentasaccharide-catalyzed rates. Although "approximation" is the predominant factor in heparin activation of ATIII thrombin inhibition, and removal of the P1 constraint plays a distinct but minor role, the primary determinant for activation of factor Xa inhibition is the pentasaccharide-induced conformational change, with approximation making a further minor contribution, and removal of the P1 constraint playing no role at all. Antithrombin III (ATIII)1 is a key inhibitor of blood coagulation enzymes and belongs to the serpin family of protease inhibitors whose members employ a common inhibition mechanism. However, the rate at which ATIII inhibits its target enzymes is abnormally low for a serpin, and activation by heparin or vascular wall heparan sulfate proteoglycans (HSPGs) is necessary for it to achieve the high inhibition rates characteristic of other serpins. The physiological importance of ATIII-heparin/HSPG interactions is supported by the occurrence of thrombosis in individuals carrying ATIII molecules with defects in heparin binding or activation, and by the clinical use of pharmaceutical heparin as an anticoagulant in patients with hereditary and acquired thrombosis.Because of the extensive use of heparin as a pharmaceutical, the mechanism for heparin activation of ATIII anticoagulant activity has been extensively investigated. These studies show that dramatically increased rates of thrombin and factor Xa inhibition can be achieved upon binding of heparin. Heparin binding to antithrombin is a two-step process consisting of an initial low affinity interaction that induces a large scale protein conformational change, which leads to high affinity binding and ATIII activation (1). The rate enhancement for the inhibition of thrombin results from an approximation (bridging) mechanism in which long, high affinity heparin chains (Ͼ18 sugars) facilitate a Ͼ1000-fold increased rate of association between thrombin and ATIII (2). In contrast, a specific anticoagulantly activ...