Residues Tyr253 and Glu255 in Strand 3 of β-Sheet C of Antithrombin Are Key Determinants of an Exosite Made Accessible by Heparin Activation to Promote Rapid Inhibition of Factors Xa and IXa

Abstract

We previously showed that conformational activation of the anticoagulant serpin, antithrombin, by heparin generates new exosites in strand 3 of ␤-sheet C, which promote the reaction of the inhibitor with the target proteases, factor Xa and factor IXa. To determine which residues comprise the exosites, we mutated strand 3C residues that are conserved in all vertebrate antithrombins. Combined mutations of the three conserved surface-accessible residues, Tyr 253 , Glu 255 , and Lys 257 , or of just Tyr 253 and Glu 255 , but not any of these residues alone, was sufficient to reproduce the exosite defects of a strand 3C antithrombin-␣ 1 -proteinase inhibitor chimera in reactions of the heparin-activated variants with both factor Xa and factor IXa. Importantly, the exosite-defective antithrombins bound heparin with nearly wild-type affinities, and the heparin-activated mutants showed near normal reactivities with thrombin, a protease that does not utilize the exosite. Mutation of the conserved but partially buried strand 3C residue, Gln 254 , the reactive loop P6 residue, Arg 399 , which interacts with Glu 255 , or a residue proposed to constitute the exosite from modeling studies, Glu 237 , all produced minimal effects on antithrombin reactivity with thrombin, factor Xa, and factor IXa in the absence or presence of heparin. Together, these results indicate that Tyr 253 and Glu 255 are key exosite determinants responsible for promoting the reactions of conformationally activated antithrombin with both factor Xa and factor IXa.Antithrombin, a member of the serpin superfamily of protein protease inhibitors, functions as a key anticoagulant regulator of blood clotting proteases in vertebrates (1). It inhibits its main target clotting proteases, thrombin, factor Xa, and factor IXa (2-4), by forming highly stable equimolar complexes with the enzymes through a novel conformational trapping mechanism (5, 6). In this mechanism, the protease recognizes and binds an exposed reactive loop of the serpin and proceeds to cleave a reactive bond in the loop to generate a covalent serpinprotease acyl-intermediate as in a regular serine protease substrate reaction. However, major conformational changes of both the serpin and protease are induced at this stage that arrest further cleavage of the bond and stabilize the acyl-intermediate complex. In these changes, the N-terminal end of the cleaved serpin-reactive loop inserts into the major ␤-sheet of the serpin core, the A sheet, dragging the acyl-linked protease along with it to the opposite end of the serpin, where steric forces distort and inactivate the protease catalytic machinery (6 -9).Antithrombin is unusual among serpins in that its reactions with its target clotting proteases are unusually slow and require the sulfated polysaccharide, heparin, to accelerate them to the physiologically significant diffusion limit. The acceleration of antithrombin-protease reactions by heparin results from the polysaccharide binding to the serpin through a unique pentasaccharide sequence and indu...

Molecular basis of factor IXa recognition by heparin-activated antithrombin revealed by a 1.7-Å structure of the ternary complex

“…Thus, contacts involving residues P6-P6′ are considered RCL contacts (1; 623-Å 2 buried surface), and those with any other part of AT are exosite contacts (921-Å 2 buried surface). The exosite interactions observed in this structure for both AT and fIXa are consistent with previous mutagenesis data (27)(28)(29) and are detailed in Tables S1 and S2. The exosite of AT is the contiguous loop 232-255, which forms strands 3 and 4 of β-sheet C and contacts the autolysis loop residues 143-153 and Glu74 in fIXa (Fig.…”

Conformational Activation of Antithrombin by Heparin Involves an Altered Exosite Interaction with Protease

“…Stoichiometry of Antithrombin-Protease Reactions-The stoichiometries of antithrombin inhibition of proteases were determined in the absence and presence of pentasaccharide and full-length heparins by end point assays essentially as described in past studies (12). Fixed concentrations of protease and heparin (ϳ100 nM) were incubated with increasing molar ratios of antithrombin to protease (up to 2:1) for a time sufficient to complete the reaction based on measured rate constants.…”

“…All mutations were confirmed by DNA sequencing. Antithrombin was expressed in Hi5 insect cells using the baculovirus expression system from Invitrogen and purified from culture media by a combination of heparin affinity and ion exchange chromatography, as previously described (12,21). Protein concentration was determined from absorbance at 280 nm using an extinction coefficient of 37,700 M Ϫ1 cm Ϫ1 (22).…”

“…In particular, the observation that mutations of a critical Tyr 253 exosite residue cause large losses in antithrombin reactivity with factors Xa and IXa in both unactivated and heparinactivated states (12) has suggested that the exosite contributes to antithrombin reactivity in both native and activated states and therefore that release of the RCL from its interaction with sheet A is not required to engage the exosite. This and other data suggested an alternative model in which the antithrombin exosite is available to interact with RCL-bound proteases in the native RCL-constrained state, but the interaction is less favorable due to repulsive interactions counteracting the attractive interactions (16).…”

“…Early models based on x-ray structures of native and heparin-activated antithrombin suggested the importance of releasing the reactive center protease binding loop (RCL) 2 from a constrained interaction with sheet A in the native state, first as a means of making the critical P1 Arg recognition determinant accessible to target proteases (11) and later as a means to gain access to exosites outside the RCL to augment the protease interaction (12)(13)(14)(15). Although a critical role for exosites in augmenting the antithrombin RCL interaction with factors Xa/IXa in the allosterically activated state is now well established, a role for RCL expulsion from sheet A in making the exosites available for interaction with protease is not supported by much available data.…”