Serpins are relatively large molecules (about 330-500 amino acids) in comparison with protease inhibitors such as basic pancreatic trypsin inhibitor (BPTI, which is about 60 amino acids) [11]. Over 70 serpin structures have been determined, and these data, along with a large amount of biochemical and biophysical information, reveal that inhibitory serpins are 'suicide' or 'single use' inhibitors that use a unique and extensive conformational change to inhibit proteases [12]. This conformational mobility renders serpins heat-labile and vulnerable to mutations that promote misfolding, spontaneous conformational change, formation of inactive serpin polymers and serpin deficiency [13]. In humans, several conformational diseases or 'serpinopathies' linked to serpin polymerization have been identified, including emphysema (SERPINA1 (antitrypsin) deficiency) [14], thrombosis (SERPINC1 (antithrombin) deficiency) [15] and angio-edema (SERPING1 (C1 esterase inhibitor) deficiency) [16]. Accumulation of serpin polymers in the endoplasmic reticulum of serpin-secreting cells can also result in disease, most notably cirrhosis (SERPINA1 polymerization) [14] and familial dementia (SERPINI1 (neuroserpin) polymerization) [17]. Other serpin-related diseases are caused by null mutations or (rarely) point mutations that alter inhibitory specificity or inhibitory function [18]. Here, we summarize the evolution, structure and mechanism of serpin function and dysfunction.

In humans, the majority (27 out of 36) of serpins are inhibitory (Table ​(Table1).1). Clade A serpins include inflammatory response molecules such as SERPINA1 (antitrypsin) and SERPINA3 (antichymotrypsin) as well as the non-inhibitory hormone-transport molecules SERPINA6 (corticosteroid-binding globulin) and SERPINA7 (thyroxine-binding globulin). Clade B includes inhibitory molecules that function to prevent inappropriate activity of cytotoxic apoptotic proteases (SERPINB6, also called PI6, and SERPINB9, also called PI9) and inhibit papain-like enzymes (SERPINB3, squamous cell carcinoma antigen-1) as well as the non-inhibitory molecule SERPINB5 (maspin). SERPINB5 does not undergo the characteristic serpin-like conformational change and functions to prevent metastasis in breast cancer and other cancers through an incompletely characterized mechanism [10,27]. The roles of several other well characterized human serpins are also summarized in Table ​Table11.

Numerous important branches of the serpin superfamily remain to be functionally characterized. For example, although plants have a large number of serpin genes, the function of plant serpins remains obscure. Studies in vitro clearly show that plant serpins can function as protease inhibitors [28], but plants lack close relatives of chymotrypsin-like proteases, which would be the obvious targets for these serpins. Thus, it has been suggested that plant serpins may be involved in inhibiting proteases in plant pathogens; for example, they may be targeting digestive proteases in insects [29]. One study convincingly demonstrated a close inverse correlation between the upregulation of Cucurbita maxima (squash) phloem serpin-1 (CmPS) and aphid survival [30]. Feeding experiments in vitro showed, however, that purified CmPS did not affect insect survival [30]. Together, these data suggest that rather than directly interacting with the pathogen, plant serpins, like their insect counterparts, may have a role in the complex pathways involved in upregulating the host immune response.

Serpins use the S-to-R transition to inhibit target proteases. Figure ​Figure1b1b shows the structure of an initial docking complex between a serpin and a protease (SERPINA1 and trypsin [35,36]) and Figure ​Figure1c1c shows the final serpin-enzyme complex [12]. These structural studies [12,35,36], combined with extensive biochemical data, revealed that RCL cleavage and subsequent insertion is crucial for effective protease inhibition. In the final serpin-protease complex, the protease remains covalently linked to the serpin, the enzyme being trapped at the acyl-intermediate stage of the catalytic cycle. Structural comparisons show that the protease in the final complex is severely distorted in comparison with the native conformation, and that much of the enzyme is disordered [12]. In addition, a fluorescence study demonstrated that the protease was partially unfolded in the final complex [37]. These conformational changes lead to distortion at the active site, which prevents efficient hydrolysis of the acyl intermediate and the subsequent release of the protease. These data are consistent with the observation that buried or cryptic cleavage sites within trypsin become exposed following complex formation with a serpin [38]. It is possible that cleavage of such cryptic sites within the protease occurs in vivo and thus results in permanent enzyme inactivation. The absolute requirement for RCL cleavage, however, means that serpins are irreversible 'suicide' inhibitors.

A major advantage of the serpin fold over small protease inhibitors such as BPTI is that the inhibitory activity of serpins can be exquisitely controlled by specific cofactors. For example, human SERPINC1 (antithrombin) is a relatively poor inhibitor of the proteases thrombin and factor Xa until it is activated by the cofactor heparin [39]. Structural studies of SERPINC1 highlight the molecular basis for heparin function. Figure ​Figure2a2a shows the structure of native SERPINC1. Here, we use the convention of Schechter and Berger, in which residues on the amino-terminal side of the cleavage site (P1/P1') are termed P2, P3, and so on, and those carboxy-terminal are termed P2', P3', and so on; corresponding subsites in the enzyme are termed S1, S2, and so on [40]. The RCL is partially inserted into the top of the 3 sheet; the residue (P1-Arg) responsible for docking into the primary specificity pocket (S1) of the protease is relatively inaccessible to docking with thrombin, as it is pointing towards and forming interactions with the body of the serpin [41,42]. Figure ​Figure2b2b illustrates the ternary complex between SERPINC1, thrombin and heparin [43]. Upon interaction with a specific heparin pentasaccharide sequence present in high-affinity heparin, SERPINC1 undergoes a substantial conformational rearrangement whereby the RCL is expelled from β-sheet A and the P1 residue flips to an exposed protease-accessible conformation [44-46]]. In addition to loop expulsion and P1 exposure, long-chain heparin can bind both enzyme and inhibitor and thus provides an additional acceleration of the inhibitory interaction. Several other serpins, including SERPIND1 (heparin cofactor II), also use cofactor binding and conformational change to achieve exquisite inhibitory control [47].

Structural studies on prokaryote and viral serpins have revealed several interesting variations of the serpin scaffold. Viral proteins are often 'stripped down' to a minimal scaffold in order to minimize the size of the viral genome. Consistent with this requirement, the structure of the viral serpin crmA, one of the smallest members of the serpin superfamily [48,49], shows that it lacks helix hD. More recently, the structure of the prokaryote serpin thermopin from Thermobifida fusca revealed the absence of helix hH [20,31]. These studies also showed that thermopin contains a 4 amino-acid insertion at the carboxyl terminus that forms extensive interactions with conserved residues at the top of β-sheet A (called the 'breach'; see later); biophysical data suggest that this region is important for proper and efficient folding of this unusual serpin.

Although the transition to latency could be an important control mechanism in at least one serpin, an alternative spontaneous conformational change, serpin polymerization, results in deficiency and disease (or serpinopathy) [14,56]. Serpin polymerization is postulated to occur via a domain-swapping event whereby the RCL of one molecule docks into β-sheet A of another to form an inactive long-chain serpin polymer (Figure 4a, b) [14,57-59]. Several important human serpin variants result in polymerization, the best studied and most common of which is the Z allele (Glu342Lys) of SERPINA1 [14]. Here, failure to properly control the activity of neutrophil elastase (the inhibitory target of SERPINA1) in the lung during the inflammatory response results in the destruction of lung tissue, leading to emphysema. Furthermore, in individuals homozygous for the Z-variant, the accumulation of serpin aggregates or polymers in the endoplasmic reticulum of anti-trypsin-producing cells, the hepatocytes, can eventually result in cell death and liver cirrhosis [14]. Similarly, mutation of SERPINI1 results in the formation of neural inclusion bodies and in the disease 'familial encephalopathy with neuroserpin inclusion bodies' (FENIB) [17,60,61].

In addition to promoting polymerization, several serpin mutations have been identified that promote formation of a disease-linked latent state. Notably, a mutation in SERPINC1, the wibble variant (Thr85Met), results in formation of large amounts of circulating latent SERPINC1 (about 10% of total SERPINC1) [55]. An alternative 'half-way house' conformation of SERPINA3, termed δ, has also been identified (Figure ​(Figure4c)4c) [62]. The structure of δ-SERPINA3 also highlights the extraordinary flexibility of the serpin scaffold: in this conformation the RCL is partially inserted into β-sheet A and helix hF has partially unwound and inserted into the base of β-sheet A, completing the β-sheet hydrogen bonding (Figure ​(Figure4c).4c). Finally, the promiscuity of β-sheet A is highlighted by the ability of this region to readily accept short peptides: several structural and biochemical studies have demonstrated that peptides can bind to β-sheet A and induce the S-to-R transition (Figure ​(Figure4d4d).

Valuable insights into the mechanism of serpin function have been gleaned from the structural location of variants that promote serpin instability [18,63]. The majority of serpinopathy-linked mutations (including antitrypsin Siiyama [64] and Mmalton [65], antithrombin wibble [55] and δ-SERPINA3 [62]) cluster in the center of the serpin molecule, underneath β-sheet A, in a region termed the shutter (marked on Figure ​Figure1a).1a). Interestingly, Glu342, the position mutated in the Z allele of SERPINA1, is located at the breach, which is just above the shutter at the top of β-sheet A. This portion of the molecule is the point of initial RCL insertion. It is suggested that destabilization of β-sheet A in either the shutter or the breach is sufficient to favor the transition to a polymeric or latent state over maintenance of the monomeric metastable native state [14]. Interestingly, analysis of conserved residues in the serpin superfamily also reveals a striking distribution of highly conserved residues stretching down the center of β-sheet A from the breach to the base of the molecule [1].

Unsurprisingly, given the important proteolytic processes they control, simple deficiencies such as those caused by null mutations of a large number of human serpins are linked to disease (some of these are summarized in Table ​Table1).1). Interestingly, however, several (rare) mutations have been identified that do not promote instability but instead interfere with the ability of the serpin to interact correctly with proteases. These include the Enschede variant of SERPINF2 [66], in which insertion of an additional alanine in the RCL results in predominantly substrate-like (rather than inhibitory) behavior upon interaction with a protease. Mutations that alter serpin specificity can also have a devastating effect. For example, the Pittsburgh variant of SERPINA1 (antitrypsin) is an effective thrombin inhibitor as a result of mutation of the P1 methionine to an arginine [67]. The carrier of this variant died of a fatal bleeding disorder in childhood.