Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation

Abstract

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10,000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin-activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins.

Figure 1

The heparin-binding mechanism of AT. The structural features of native and pentasaccharide-activated AT are illustrated by ribbon diagrams. AT is shown in the classic orientation, with β-sheet A (blue) facing, helix A (green) in the back, the principal heparin-binding helix D (cyan) to the right, and the RCL (yellow) at the top. RCL residues are numbered from the scissile P1–P1′ bond towards the N- and C-termini, respectively, and the positions of the P4 and P3′ residues are indicated. In native AT the N-terminal region of the RCL (hinge region, circle) is incorporated as strand 4 in β-sheet A, which constrains the RCL and the P1 Arg393 side chain (red space-filling). The heparin pentasaccharide (rods with gray C, red O, and yellow S) binds with contacts on helices A and D and induces local and global conformational changes in AT. Of particular relevance is the expulsion of the hinge region from β-sheet A, which predictably releases the constraints on the RCL and reorients the P1 side chain.

Figure 2

Crystal structure of the Michaelis complex between pentasaccharide-activated AT and fXa. (A) Stereo view of the complex with AT colored as before, the protease domain of fXa in magenta, and the second EGF-like domain of fXa in purple (with disulfide bonds shown in green). The orientation of fXa on AT is slightly to the left of the long axis of AT, and is rotated significantly to the left to engage in direct exosite contacts with the top surface of AT. The positions of the AT mutations which allowed crystal contacts to form are indicated by orange balls. (B) It is clear from the stereo representation of the AT–thrombin Michaelis complex superimposed on the AT–fXa complex why one is insensitive to hinge region expulsion and the other critically depends on hinge region extension (hinge region is circled). The RCL of the thrombin complex is orange, with green rods for P4 Ile390 and P1 Arg393, and the RCL from the fXa complex is yellow, with P4 and P1 residues colored red. Thrombin (semitransparent cyan) is oriented towards the front of AT and the RCL enters the active site of thrombin by the most direct line, with P4–P1 aligned along the z-axis (into the plane). In contrast, fXa is translated towards the left and rotated so that the P4–P1 enters the active site cleft along the x-axis (from right to left). The position and orientation of the active site cleft of fXa is dictated by the exosite contacts, which are evident to the left of the RCL. The forward position and orientation of thrombin permit short heparin chains (∼16mer) to bridge the heparin-binding sites of the two molecules (semitransparent cyan rods from the structure by Li et al, 2004). In contrast, based on the position and orientation of fXa observed here, a 28mer (magenta rods for the modeled heparin chain) would be predicted to be the minimal required heparin length for bridging the AT–fXa Michaelis complex.

Figure 3

The AT–pentasaccharide interactions. (A) A stereo representation of the heparin-binding region of AT (colored as before) with 2Fo−Fc electron density contoured at one times the RMSD of the map (1σ). All side chains which interact directly with the pentasaccharide (fondaparinux) are shown as rods, with carbon atoms colored as the helix on which they are found (green for helix A and cyan for helix D). (B) Direct side-chain interactions are illustrated schematically, with salt bridges indicated by solid lines and hydrogen bonds indicated by dashed lines. Individual monosaccharide units are traditionally labeled from D to H. An exhaustive list of all interactions is given in Supplementary Table 1.

Figure 4

Stereo views of the interactions between AT (yellow) and fXa (magenta). (A) Interactions between the RCL (P4 I390–P2′ L395) of AT and active site residues of fXa are shown, with 2Fo−Fc electron density surrounding the RCL contoured at 1σ. The interactions are indicated by broken green lines and are generally sequence nonspecific, with the exception of the extensive P1 Arg393 side-chain interactions. (B) The first exosite on fXa includes residues from the 36-loop, from Asn35 to Phe41, and involves two important salt bridges. (C) The most important exosite on fXa is the autolysis loop, from Arg143 to Gln151. The side chain of Arg150 participates in a salt bridge with Glu237 and is neatly sandwiched between the side chains of Arg235 and Tyr253.

Figure 5

Stereo views of the contact surfaces on fXa and AT. fXa and AT surfaces within 4.5 Å of one another are colored green to illustrate the extent of the active site and exosite contacts, and contacting residues are depicted as rods. (A) The surface of fXa is shown in the standard orientation (active site facing with substrate running from left to right, from N- to C-terminus). RCL residues P4–P2′ are represented as white rods (P3′ is not shown because it makes no contact), and exosite-contacting AT residues are shown in yellow. The two exosites on fXa are indicated by the ovals; exosite 1 contains the 36-loop, and exosite 2 is the autolysis loop. (B) The top surface of AT with the RCL removed is colored as above, with contacting residues from fXa colored magenta. The oval indicates the position of the 36-loop contacts and the square indicates the position of the autolysis loop contacts. The AT residues under the oval derive from the RCL C-terminal insertion loop, s1C, and s1B, and those under the square derive from s3C and s4C.

Figure 6

Holes and knobs that form the principal interaction sites are approximated by pentasaccharide activation. The two principal interactions between arginine side chains and corresponding acidic pockets are illustrated by coloring surfaces according to electrostatics (red for negative and blue for positive). (A) The P1 Arg393 of AT is buried deep in the S1 pocket (233 Ų buried) of fXa, and its interactions are indicated by dashed yellow lines. (B) The principal exosite contact involves a similar burying of Arg150 from fXa into an acidic pocket on the surface between strands 3 and 4 of β-sheet C (194 Ų buried). (C) A schematic representation is shown to illustrate the hypothesis of allosteric activation of AT towards fXa. Each molecule possesses a hole and a knob, which can interact individually but not simultaneously until native AT (nAT) binds to the specific heparin pentasaccharide (Penta). The resulting conformational change does not necessarily improve the accessibility of the hole and knob on AT, but rather repositions the knob (Arg393) to allow its engagement in the S1 pocket and the simultaneous engagement of the exosite knob of Arg150. The repositioning of Arg393 is only possible when the hinge region is freed from β-sheet A (see Figure 1).