James A. Huntington

Structure of a serpin-protease complex shows inhibition by deformation.

The serpins have evolved to be the predominant family of serine-protease inhibitors in man. Their unique mechanism of inhibition involves a profound change in conformation, although the nature and significance of this change has been controversial. Here we report the crystallographic structure of a typical serpin–protease complex and show the mechanism of inhibition. The conformational change is initiated by reaction of the active serine of the protease with the reactive centre of the serpin. This cleaves the reactive centre, which then moves 71 Å to the opposite pole of the serpin, taking the tethered protease with it. The tight linkage of the two molecules and resulting overlap of their structures does not affect the hyperstable serpin, but causes a surprising 37% loss of structure in the protease. This is induced by the plucking of the serine from its active site, together with breakage of interactions formed during zymogen activation. The disruption of the catalytic site prevents the release of the protease from the complex, and the structural disorder allows its proteolytic destruction. It is this ability of the conformational mechanism to crush as well as inhibit proteases that provides the serpins with their selective advantage.

Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin.

The maintenance of normal blood flow depends completely on the inhibition of thrombin by antithrombin, a member of the serpin family. Antithrombin circulates at a high concentration, but only becomes capable of efficient thrombin inhibition on interaction with heparin or related glycosaminoglycans. The anticoagulant properties of therapeutic heparin are mediated by its interaction with antithrombin, although the structural basis for this interaction is unclear. Here we present the crystal structure at a resolution of 2.5 Å of the ternary complex between antithrombin, thrombin and a heparin mimetic (SR123781). The structure reveals a template mechanism with antithrombin and thrombin bound to the same heparin chain. A notably close contact interface, comprised of extensive active site and exosite interactions, explains, in molecular detail, the basis of the antithrombotic properties of therapeutic heparin.

Serpins in thrombosis, hemostasis and fibrinolysis.

Summary.  Hemostasis and fibrinolysis, the biological processes that maintain proper blood flow, are the consequence of a complex series of cascading enzymatic reactions. Serine proteases involved in these processes are regulated by feedback loops, local cofactor molecules, and serine protease inhibitors (serpins). The delicate balance between proteolytic and inhibitory reactions in hemostasis and fibrinolysis, described by the coagulation, protein C and fibrinolytic pathways, can be disrupted, resulting in the pathological conditions of thrombosis or abnormal bleeding. Medicine capitalizes on the importance of serpins, using therapeutics to manipulate the serpin–protease reactions for the treatment and prevention of thrombosis and hemorrhage. Therefore, investigation of serpins, their cofactors, and their structure–function relationships is imperative for the development of state‐of‐the‐art pharmaceuticals for the selective fine‐tuning of hemostasis and fibrinolysis. This review describes key serpins important in the regulation of these pathways: antithrombin, heparin cofactor II, protein Z‐dependent protease inhibitor, α1‐protease inhibitor, protein C inhibitor, α2‐antiplasmin and plasminogen activator inhibitor‐1. We focus on the biological function, the important structural elements, their known non‐hemostatic roles, the pathologies related to deficiencies or dysfunction, and the therapeutic roles of specific serpins.

Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization

Repeating intermolecular protein association by means of β-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer’s, Huntington’s and Parkinson’s and the prion encephalopathies. A family of proteins, known as the serpins, also forms large stable multimers by ordered β-sheet linkages leading to intracellular accretion and disease. These ‘serpinopathies’ include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in α1-antitrypsin (α1AT), and thrombosis caused by mutations in antithrombin. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the β-sheet expansion disorders collectively known as the conformational diseases. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel β-strands inserting in the centre of the principal β-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible β-sheet expansion.

How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration

The interaction of the plasma protein vitronectin with plasminogen activator inhibitor-1 (PAI-1) is central to human health. Vitronectin binding extends the lifetime of active PAI-1, which controls hemostasis by inhibiting fibrinolysis and has also been implicated in angiogenesis. The PAI-1–vitronectin binding interaction also affects cell adhesion and motility. For these reasons, elevated PAI-1 activities are associated both with coronary thrombosis and with a poor prognosis in many cancers. Here we show the crystal structure at a resolution of 2.3 Å of the complex of the somatomedin B domain of vitronectin with PAI-1. The structure of the complex explains how vitronectin binds to and stabilizes the active conformation of PAI-1. It also explains the tissue effects of PAI-1, as PAI-1 competes for and sterically blocks the interaction of vitronectin with cell surface receptors and integrins. Structural understanding of the essential biological roles of the interaction between PAI-1 and vitronectin opens the prospect of specifically designed blocking agents for the prevention of thrombosis and treatment of cancer.

Molecular recognition mechanisms of thrombin

Summary.  Thrombin is the final protease generated in the blood coagulation cascade, and is the only factor capable of cleaving fibrinogen to create a fibrin clot. Unlike every other coagulation protease, thrombin is composed solely of its serine protease domain, so that once formed it can diffuse freely to encounter a large number of potential substrates. Thus thrombin serves many functions in hemostasis through the specific cleavage of at least a dozen substrates. The solution of the crystal structure of thrombin some 15 years ago revealed a deep active site cleft and two adjacent basic exosites, and it was clear that thrombin must utilize these unique features in recognizing its substrates. Just how this occurs is still being investigated, but recent data from thrombin mutant libraries and crystal structures combine to paint the clearest picture to date of the molecular determinants of substrate recognition by thrombin. In almost all cases, both thrombin exosites are involved, either through direct interaction with the substrate protein or through indirect interaction with a third cofactor molecule. The purpose of this article is to summarize recent biochemical and structural data in order to provide insight into the thrombin molecular recognition events at the heart of hemostasis.

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

The serine proteases sequentially activated to form a fibrin clot are inhibited primarily by members of the serpin family, which use a unique β-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 β-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 β-sheet of HCII.

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

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.

Serpin structure, function and dysfunction.

Summary.  Serpins have been studied as a distinct protein superfamily since the early 80s. In spite of the poor sequence homology between family members, serpins share a highly conserved core structure that is critical for their functioning as serine protease inhibitors. Therefore, discoveries made about one serpin can be related to the others. In this short review, I introduce the serpin structure and general mechanism of protease inhibition, and illustrate, using recent crystallographic and biochemical data on antithrombin (AT), how serpin activity can be modulated by cofactors. The ability of the serpins to undergo conformational change is critical for their function, but it also renders them uniquely susceptible to mutations that perturb their folding, leading to deficiency and disease. A recent crystal structure of an AT dimer revealed that serpins can participate in large‐scale domain‐swaps to form stable polymers, and that such a mechanism may explain the accumulation of misfolded serpins within secretory cells. Serpins play important roles in haemostasis and fibrinolysis, and although each will have some elements specifically tailored for its individual function, the mechanisms described here provide a general conceptual framework.

Mechanisms of glycosaminoglycan activation of the serpins in hemostasis

Summary.  Serpins are the predominant protease inhibitors in the higher organisms and are responsible, in humans, for the control of many highly regulated processes including blood coagulation and fibrinolysis. The serpin inhibitory mechanism has recently been revealed by the solution of a crystallographic structure of the final serpin–protease complex. The serpin mechanism, in contrast to the classical lock‐and‐key mechanism, involves dramatic conformational change in both the inhibitor and the inhibited protein. The final result is a stable covalent complex in which the properties of each component are altered so as to allow clearance from the circulation. Several serpins are involved in hemostasis: antithrombin (AT) inhibits many coagulation proteases, most importantly factor Xa and thrombin; heparin cofactor II (HCII) inhibits thrombin; protein C inhibitor (PCI) inhibits activated protein C and thrombin bound to thrombomodulin; plasminogen activator inhibitor 1 inhibits tissue plasminogen activator; and α2‐antiplasmin inhibits plasmin. Nearly all of these reactions are accelerated through interactions with glycosaminoglycans (GAGs) such as heparin or heparan sulfate. Recent structures of AT, HCII and PCI have revealed how in each case the serpin mechanism has been fine‐tuned by evolution to bring about high levels of regulatory control, and how seemingly disparate mechanisms of GAG binding and activation can share critical elements. By considering the serpins involved in hemostasis together it is possible to develop a deeper understanding of their complex individual roles.