Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism

Abstract

The serine proteases sequentially activated to form a fibrin clot are inhibited primarily by members of the serpin family, which use a unique ␤-sheet expansion mechanism to trap and destroy their targets. Since the discovery that serpins were a family of serine protease inhibitors there has been controversy as to the role of conformational change in their mechanism. It now is clear that protease inhibition depends entirely on rapid serpin ␤-sheet expansion after proteolytic attack. The regulatory advantage afforded by the conformational mobility of serpins is demonstrated here by the structures of native and S195A thrombin-complexed heparin cofactor II (HCII). HCII inhibits thrombin, the final protease of the coagulation cascade, in a glycosaminoglycan-dependent manner that involves the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. The native structure of HCII resembles that of native antithrombin and suggests an alternative mechanism of allosteric activation, whereas the structure of the S195A thrombin-HCII complex defines the molecular basis of allostery. Together, these structures reveal a multistep allosteric mechanism that relies on sequential contraction and expansion of the central ␤-sheet of HCII.T he predominant protease inhibitors of the higher organisms, the serpins (1, 2), have evolved a complex mechanism that was revealed recently by the crystallographic structure of a serpin-protease complex (3). This dramatic mechanism amounts to a race between proteolysis and the incorporation of the serpin-reactive center loop into its main ␤-sheet (␤-sheet A). The dependence of the serpin inhibitory mechanism on ␤-sheet expansion renders serpins highly susceptible to both loss-offunction and gain-of-function diseases but additionally allows for a range of mechanisms for the modulation of serpin activity. Thus, serpins typically are found controlling tightly regulated physiological pathways such as blood coagulation. Antithrombin (AT) and heparin cofactor II (HCII) have independently (4, 5) evolved mechanisms by which they circulate in an inert state until activated by binding to cell surface glycosaminoglycans (GAGs). AT and HCII thus are allosterically activated toward factor Xa and thrombin, respectively, the final two proteases in the blood coagulation cascade.The structure and mechanism of activation of AT has been well characterized. Its fold is similar to that of the rest of the serpin family with the single and important exception that the reactive center loop is constrained through partial incorporation into ␤-sheet A (refs. 6 and 7; Fig. 1a). The native conformation of AT thus renders it incapable of forming a productive recognition complex (Michaelis complex) with factor Xa, and only as the result the conformational changes brought about through the interaction with a specific heparin pentasaccharide sequence does AT become an efficient inhibitor of factor Xa. Pentasaccharide binding results in secondary structural changes in the heparin binding region and the ex...

Proteolytic Activation Transforms Heparin Cofactor II into a Host Defense Molecule

“…These two helices are both cationic and amphipathic (6), which are characteristics shared with many classical antimicrobial and LPS-interacting peptides such as LL-37 (22). Therefore, the current study implicates that the exposure of these two regions in the elongated HCIIa form underlies the binding of HCIIa to LPS and bacteria and its functional implications for bacterial clearance.…”

“…HCII has two heparin-binding regions, located to the cationic and amphipathic helices A and D (6). To further define the epitopes responsible for the LPS-binding and antibacterial actions of intact HCIIa, peptides comprising those two helices, but also the other exposed helical regions of HCII (marked by different colors in Fig.…”

“…In contrast to ATIII, however, HCII exploits its conformational mobility in a unique way. HCII has an unusual N-terminal acidic region, which, upon binding of HCII to glycosaminoglycans, is expelled from the molecules' cationic site, followed by proteolytic scission of the flexible N-terminal tail (6). Proteolysis of HCII by leukocyte elastase subsequently releases chemoattractant peptides derived from the N-terminal tail (7).…”

A Hexasaccharide Containing Rare 2‐O‐Sulfate‐Glucuronic Acid Residues Selectively Activates Heparin Cofactor II

“…Yet, whereas AT binds specifically to fondaparinux sequence in heparin,7 HCII is considered to bind non‐specifically to heparin and HS. Further, although both serpins display a two‐step, induced‐fit, allosteric activation mechanism in inhibiting their target enzymes,8, 9, 10 no HS oligosaccharide has been discovered to induce robust activation of HCII.…”

“…To identify HCII‐specific sequences, we compared the overall tertiary structures of HCII and AT 7, 9. The HS binding site in AT is formed by helices A (hA) and D (hD), which contains R46, R47, K114, K125, R129, R132 and K133 that define the specificity of HS recognition.…”