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Serpins are a superfamily of with similar structures that were first identified for their protease inhibition activity and are found in all kingdoms of life. The acronym serpin was originally coined because the first serpins to be identified act on chymotrypsin-like ( serine protease inhibitors). (1986). 9780444807632, Elsevier Science Publishers BV. ISBN 9780444807632 They are notable for their unusual mechanism of action, in which they irreversibly inhibit their target protease by undergoing a large conformational change to disrupt the target's active site. This contrasts with the more common competitive mechanism for protease inhibitors that bind to and block access to the protease active site.
Protease inhibition by serpins controls an array of biological processes, including coagulation and inflammation, and consequently these proteins are the target of medical research. Their unique conformational change also makes them of interest to the structural biology and protein folding research communities. The conformational-change mechanism confers certain advantages, but it also has drawbacks: serpins are vulnerable to mutations that can result in serpinopathies such as Proteopathy and the formation of inactive long-chain . Serpin polymerisation not only reduces the amount of active inhibitor, but also leads to accumulation of the polymers, causing cell death and organ failure.
Although most serpins control proteolysis cascades, some proteins with a serpin structure are not , but instead perform diverse functions such as storage protein (as in egg white—ovalbumin), transport as in hormone carriage proteins (thyroxine-binding globulin, transcortin) and molecular chaperoning (HSP47). The term serpin is used to describe these members as well, despite their non-inhibitory function, since they are evolutionarily related.
The critical role of the active centre residue in determining the specificity of inhibition of serpins was unequivocally confirmed by the finding that a natural mutation of the active centre methionine in alpha1-antitrypsin to an arginine, as in antithrombin, resulted in a severe bleeding disorder. This active-centre specificity of inhibition was also evident in the many other families of protease inhibitors but the serpins differed from them in being much larger proteins and also in possessing what was soon apparent as an inherent ability to undergo a change in shape. The nature of this conformational change was revealed with the determination in 1984 of the first crystal structure of a serpin, that of post-cleavage alpha1-antitrypsin. This together with the subsequent solving of the structure of native (uncleaved) ovalbumin indicated that the inhibitory mechanism of the serpins involved a remarkable conformational shift, with the movement of the exposed peptide loop containing the reactive site and its incorporation as a middle strand in the main beta-pleated sheet that characterises the serpin molecule. Early evidence of the essential role of this loop movement in the inhibitory mechanism came from the finding that even minor aberrations in the amino acid residues that form the hinge of the movement in antithrombin resulted in thrombotic disease. Ultimate confirmation of the linked displacement of the target protease by this loop movement was provided in 2000 by the structure of the post-inhibitory complex of alpha1-antitrypsin with trypsin, showing how the displacement results in the deformation and inactivation of the attached protease. Subsequent structural studies have revealed an additional advantage of the conformational mechanism in allowing the subtle modulation of inhibitory activity, as notably seen at tissue level with the functionally diverse serpins in human plasma.
Over 1000 serpins have now been identified, including 36 human proteins, as well as molecules in all kingdoms of life—animals, plants, fungi, bacteria, and archaea—and some Poxviridae. The central feature of all is a tightly conserved framework, which allows the precise alignment of their key structural and functional components based on the template structure of alpha1-antitrypsin. In the 2000s, a systematic nomenclature was introduced in order to categorise members of the serpin superfamily based on their evolutionary relationships. Serpins are therefore the largest and most diverse superfamily of protease inhibitors.
Some serpins inhibit other protease classes, typically cysteine proteases, and are termed "cross-class inhibitors". These enzymes differ from serine proteases in that they use a nucleophilic cysteine residue, rather than a serine, in their active site. Nonetheless, the enzymatic chemistry is similar, and the mechanism of inhibition by serpins is the same for both classes of protease. Examples of cross-class inhibitory serpins include serpin B4 a squamous cell carcinoma antigen 1 (SCCA-1) and the avian serpin myeloid and erythroid nuclear termination stage-specific protein (MENT), which both inhibit papain-like cysteine proteases.
The protease targets of intracellular inhibitory serpins have been difficult to identify, since many of these molecules appear to perform overlapping roles. Further, many human serpins lack precise functional equivalents in model organisms such as the mouse. Nevertheless, an important function of intracellular serpins may be to protect against the inappropriate activity of proteases inside the cell. For example, one of the best-characterised human intracellular serpins is Serpin B9, which inhibits the cytotoxic granule protease granzyme B. In doing so, Serpin B9 may protect against inadvertent release of granzyme B and premature or unwanted activation of apoptosis pathways.
Some use serpins to disrupt protease functions in their host. The cowpox viral protein CrmA (cytokine response modifier A) is used in order to avoid inflammatory and apoptosis responses of infected host cells. CrmA increases infectivity by suppressing its host's inflammatory response through inhibition of IL-1 and IL-18 processing by the cysteine protease caspase-1. In eukaryotes, a plant serpin inhibits both and a papain-like cysteine protease.
Some serpins are both protease inhibitors and perform additional roles. For example, the nuclear cysteine protease inhibitor MENT, in also acts as a chromatin remodelling molecule in a bird's red blood cells.
The serpin structures that have been determined cover several different conformations, which has been necessary for the understanding of their multiple-step mechanism of action. Structural biology has therefore played a central role in the understanding of serpin function and biology.
Serine protease and cysteine proteases catalyse peptide bond cleavage by a two-step process. Initially, the catalytic residue of the active site Catalytic triad performs a nucleophile attack on the peptide bond of the substrate. This releases the new N-terminus and forms a covalent ester-bond between the enzyme and the substrate. This covalent complex between enzyme and substrate is called an acyl-enzyme intermediate. For standard substrates, the ester bond is Hydrolysis and the new C-terminus is released to complete catalysis. However, when a serpin is cleaved by a protease, it rapidly undergoes the S to R transition before the acyl-enzyme intermediate is hydrolysed. The efficiency of inhibition depends on fact that the relative Enzyme kinetics of the conformational change is several orders of magnitude faster than hydrolysis by the protease.
Since the RCL is still covalently attached to the protease via the ester bond, the S to R transition pulls protease from the top to the bottom of the serpin and distorts the catalytic triad. The distorted protease can only hydrolyse the acyl enzyme intermediate extremely slowly and so the protease remains covalently attached for days to weeks. Serpins are classed as irreversible inhibitors and as suicide inhibitors since each serpin protein permanently inactivates a single protease, and can only function once.
The archetypal example of this situation is antithrombin, which circulates in plasma in a partially inserted relatively inactive state. The primary specificity determining residue (the P1 arginine) points toward the body of the serpin and is unavailable to the protease. Upon binding a high-affinity pentasaccharide sequence within long-chain heparin, antithrombin undergoes a conformational change, RCL expulsion, and exposure of the P1 arginine. The heparin pentasaccharide-bound form of antithrombin is, thus, a more effective inhibitor of thrombin and factor Xa. Furthermore, both of these coagulation proteases also contain binding sites (called exosites) for heparin. Heparin, therefore, also acts as a template for binding of both protease and serpin, further dramatically accelerating the interaction between the two parties. After the initial interaction, the final serpin complex is formed and the heparin moiety is released. This interaction is physiologically important. For example, after injury to the blood vessel wall, heparin is exposed, and antithrombin is activated to control the clotting response. Understanding of the molecular basis of this interaction enabled the development of Fondaparinux, a synthetic form of Heparin pentasaccharide used as an anticoagulant.
Regulation of the latency transition can act as a control mechanism in some serpins, such as PAI-1. Although PAI-1 is produced in the inhibitory S conformation, it "auto-inactivates" by changing to the latent state unless it is bound to the cofactor vitronectin. Similarly, antithrombin can also spontaneously convert to the latent state, as an additional modulation mechanism to its allosteric activation by heparin. Finally, the N-terminus of , a serpin from Thermoanaerobacter tengcongensis, is required to lock the molecule in the native inhibitory state. Disruption of interactions made by the N-terminal region results in spontaneous conformational change of this serpin to the latent conformation.
In some serpins, the S to R transition can activate Cell signaling events. In these cases, a serpin that has formed a complex with its target protease, is then recognised by a receptor. The binding event then leads to downstream signalling by the receptor. The S to R transition is therefore used to alert cells to the presence of protease activity. This differs from the usual mechanism whereby serpins affect signalling simply by inhibiting proteases involved in a signalling cascade.
Mutations that affect the rate or the extent of RCL insertion into the A-sheet can cause the serpin to undergo its S to R conformational change before having engaged a protease. Since a serpin can only make this conformational change once, the resulting misfired serpin is inactive and unable to properly control its target protease. Similarly, mutations that promote inappropriate transition to the monomeric latent state cause disease by reducing the amount of active inhibitory serpin. For example, the disease-linked antithrombin variants wibble and wobble, both promote formation of the latent state.
The structure of the disease-linked mutant of antichymotrypsin (L55P) revealed another, inactive "δ-conformation". In the δ-conformation, four residues of the RCL are inserted into the top of β-sheet A. The bottom half of the sheet is filled as a result of one of the α-helices (the F-helix) partially switching to a β-strand conformation, completing the β-sheet hydrogen bonding. It is unclear whether other serpins can adopt this conformer, and whether this conformation has a functional role, but it is speculated that the δ-conformation may be adopted by Thyroxine-binding globulin during thyroxine release. The non-inhibitory proteins related to serpins can also cause diseases when mutated. For example, mutations in SERPINF1 cause osteogenesis imperfecta type VI in humans.
In the absence of a required serpin, the protease that it normally would regulate is over-active, leading to pathologies. Consequently, simple deficiency of a serpin (e.g. a null mutation) can result in disease. , particularly in Knockout mouse, are used experimentally to determine the normal functions of serpins by the effect of their absence.
Each monomer of the serpin aggregate exists in the inactive, relaxed conformation (with the RCL inserted into the A-sheet). The polymers are therefore hyperstable to temperature and unable to inhibit proteases. Serpinopathies therefore cause pathologies similarly to other proteopathy (e.g. prion diseases) via two main mechanisms. First, the lack of active serpin results in uncontrolled protease activity and tissue destruction. Second, the hyperstable polymers themselves clog up the endoplasmic reticulum of cells that synthesize serpins, eventually resulting in cell death and tissue damage. In the case of antitrypsin deficiency, antitrypsin polymers cause the death of Hepatocyte, sometimes resulting in liver damage and cirrhosis. Within the cell, serpin polymers are slowly removed via degradation in the endoplasmic reticulum. However, the details of how serpin polymers cause cell death remains to be fully understood.
Physiological serpin polymers are thought to form via Protein domain events, where a segment of one serpin protein inserts into another. Domain-swaps occur when mutations or environmental factors interfere with the final stages of serpin folding to the native state, causing high-energy intermediates to misfold. Both protein dimer and protein trimer domain-swap structures have been solved. In the dimer (of antithrombin), the RCL and part of the A-sheet incorporates into the A-sheet of another serpin molecule. The domain-swapped trimer (of antitrypsin) forms via the exchange of an entirely different region of the structure, the B-sheet (with each molecule's RCL inserted into its own A-sheet). It has also been proposed that serpins may form domain-swaps by inserting the RCL of one protein into the A-sheet of another (A-sheet polymerisation). These domain-swapped dimer and trimer structures are thought to be the building blocks of the disease-causing polymer aggregates, but the exact mechanism is still unclear.
Protease-inhibition is thought to be the ancestral function, with non-inhibitory members the results of evolutionary neofunctionalisation of the structure. The S to R conformational change has also been adapted by some binding serpins to regulate affinity for their targets.
| Table of human serpins | |||||||
| SERPINA1 | α1-antitrypsin | Extracellular | Inhibitor of human neutrophil elastase. The C-terminal fragment of cleaved SERPINA1 may inhibit HIV-1 infection. | Deficiency results in emphysema, polymerisation results in cirrhosis (serpinopathy). | 14q32.1 | , , | |
| SERPINA2 | Antitrypsin-related protein | Extracellular | Possible pseudogene. | 14q32.1 | |||
| SERPINA3 | α1-antichymotrypsin | Extracellular | Inhibitor of cathepsin G. Additional roles in chromatin condensation in hepatic cells. | Mis-regulation results in Alzheimer's disease (serpinopathy). | 14q32.1 | , | |
| SERPINA4 | Kallistatin | Extracellular | Inhibitor of kallikrein, regulator of vascular function. | Depletion in hypertensive rats exacerbates renal and cardiovascular injury. | 14q32.1 | ||
| SERPINA5 | Protein C inhibitor | Extracellular | Inhibitor of active protein C. Intracellular role in preventing phagocytosis of bacteria. | Knockout in male mice causes infertility. Accumulation occurs in chronic active plaques in multiple sclerosis. | 14q32.1 | , | |
| SERPINA6 | Transcortin | Extracellular | Non-inhibitory. Cortisol binding. | Deficiency associated with chronic fatigue. | 14q32.1 | , , | |
| SERPINA7 | Thyroxine-binding globulin | Extracellular | Non-inhibitory. Thyroxine binding. | Deficiency causes hypothyroidism. | Xq22.2 | , , | |
| SERPINA8 | Angiotensinogen | Extracellular | Non-inhibitory, cleavage by renin results in release of angiotensin I. | Knockout in mice causes hypotension. | Variants linked to hypertension. | 1q42-q43 | , , , , , , |
| SERPINA9 | Centerin / GCET1 | Extracellular | Inhibitory, maintenance of naive B cells. | Strongly expressed in most B-cell lymphomas. | 14q32.1 | ||
| SERPINA10 | Protein Z-related protease inhibitor | Extracellular | Binds protein Z and inactivates factor Xa and . | 14q32.1 | , | ||
| SERPINA11 | – | Probably extracellular | Unknown | 14q32.13 | |||
| SERPINA12 | Vaspin | Extracellular | Inhibitor of Kallikrein-7. Insulin-sensitizing adipocytokine. | High plasma levels associated with type II diabetes. | 14q32.1 | ||
| SERPINA13 | – | Probably extracellular | Unknown | 14q32 | |||
| SERPINB1 | Monocyte neutrophil elastase inhibitor | Intracellular | Inhibitor of neutrophil elastase. | Knockout in mice causes neutrophil survival defect and immune deficiency. | 6p25 | ||
| SERPINB2 | Plasminogen activator inhibitor-2 | Intracellular/extracellular | Inhibitor of extracellular uPA. Intracellular function unclear, but may protect against viral infection. | Deficiency in mice reduces immune response to nematode infection. Knockout in mice causes no obvious phenotype. | 18q21.3 | ||
| SERPINB3 | Squamous cell carcinoma antigen-1 (SCCA-1) | Intracellular | Inhibitor of papain-like cysteine proteases and cathepsins K, L and S. | Knockout in mice of Serpinb3a (the murine homolog of both human SERPINB3 and SERPINB4) have reduced mucus production in a murine model of asthma. | 18q21.3 | ||
| SERPINB4 | Squamous cell carcinoma antigen-2 (SCCA-2) | Intracellular | Inhibitor of chymotrypsin-like serine proteases, cathepsin G and chymase. | Knockout in mice of Serpinb3a (the murine homolog of both human SERPINB3 and SERPINB4) have reduced mucus production in a murine model of asthma. | 18q21.3 | ||
| SERPINB5 | Maspin | Intracellular | Non-inhibitory, function unclear ( see also maspin) | Knockout in mice originally reported as lethal, but subsequently shown to have no obvious phenotype. Expression may be a prognostic indicator that reflects expression of a neighbouring tumour suppressor gene (the phosphatase PHLPP1). | 18q21.3 | ||
| SERPINB6 | PI-6 | Intracellular | Inhibitor of cathepsin G. | Knockout in mice causes hearing loss and mild neutropenia. | Deficiency associated with hearing loss. | 6p25 | |
| SERPINB7 | Megsin | Intracellular | Involved in megakaryocyte maturation. | Over-expression in mice causes kidney disease. Knockout in mice does not cause histological abnormalities. | Mutations associated with Nagashima-type Palmoplantar Keratosis. | 18q21.3 | |
| SERPINB8 | PI-8 | Intracellular | Possible inhibitor of furin. | 18q21.3 | |||
| SERPINB9 | PI-9 | Intracellular | Inhibitor of the cytotoxic granule protease granzyme B. | Knockout in mice causes immune dysfunction. | 6p25 | ||
| SERPINB10 | Bomapin | Intracellular | Unknown | Knockout in mice causes no obvious phenotype (C57/BL6; lab strain BC069938). | 18q21.3 | ||
| SERPINB11 | Intracellular | Unknown | Murine Serpinb11 is an active inhibitor whereas the human orthalogue is inactive. Deficiency in ponies is associated with hoof wall separation disease. | 18q21.3 | |||
| SERPINB12 | Yukopin | Intracellular | Unknown | 18q21.3 | |||
| SERPINB13 | Hurpin/Headpin | Intracellular | Inhibitor of papain-like cysteine proteases. | 18q21.3 | |||
| SERPINC1 | Antithrombin | Extracellular | Inhibitor of coagulation, specifically factor X, factor IX and thrombin. | Knockouts in mice are lethal. | Deficiency results in thrombosis and other clotting disorders (serpinopathy). | 1q23-q21 | , , , , , |
| SERPIND1 | Heparin cofactor II | Extracellular | Inhibitor of thrombin. | Knockouts in mice are lethal. | 22q11 | , | |
| SERPINE1 | Plasminogen activator inhibitor 1 | Extracellular | Inhibitor of thrombin, uPA and TPa. | 7q21.3-q22 | , | ||
| SERPINE2 | Glia derived nexin / Protease nexin I | Extracellular | Inhibitor of uPA and tPA. | Abnormal expression leads to male infertility. Knockout in mice causes epilepsy. | 2q33-q35 | ||
| SERPINF1 | Pigment epithelium derived factor | Extracellular | Non-inhibitory, potent anti-angiogenic molecule. PEDF has been reported to bind the glycosaminoglycan hyaluronan. | Knockout in mice affects the vasculature and mass of the pancreas and the prostate. Promotes Notch–dependent renewal of adult periventricular neural stem cells. Mutations in humans cause osteogenesis imperfecta type VI. | 17p13.3 | ||
| SERPINF2 | α2-antiplasmin | Extracellular | Inhibitor of plasmin, inhibitor of fibrinolysis. | Knockouts in mice show increased mice show increased fibrinolysis but no bleeding disorder. | Deficiency causes a rare bleeding disorder. | 17pter-p12 | |
| SERPING1 | Complement 1-inhibitor | Extracellular | Inhibitor of C1 esterase. | Several polymorphisms associated with macular degeneration and hereditary angeoedema. | 11q11-q13.1 | ||
| SERPINH1 | 47 kDa Heat shock protein (HSP47) | Intracellular | Non-inhibitory, molecular chaperone in collagen folding. | Knockouts in mice are lethal. | Mutation in humans causes severe osteogenesis imperfecta. | 11p15 | |
| SERPINI1 | Neuroserpin | Extracellular | Inhibitor of tPA, uPA and plasmin. | Mutation causes FENIB dementia (serpinopathy). | 3q26 | , , , | |
| SERPINI2 | Pancpin | Extracellular | Unknown | Deficiency in mice causes pancreatic insufficiency via acinar cell loss. | 3q26 |
Studies on Drosophila serpins reveal that Serpin-27A inhibits the Easter protease (the final protease in the Nudel, Gastrulation Defective, Snake and Easter proteolytic cascade) and thus controls dorsoventral patterning. Easter functions to cleave Spätzle (a chemokine-type ligand), which results in toll-mediated signaling. As well as its central role in embryonic patterning, toll signaling is also important for the innate immune response in insects. Accordingly, serpin-27A also functions to control the insect immune response. In Tenebrio molitor (a large beetle), a protein (SPN93) comprising two discrete tandem serpin domains functions to regulate the toll proteolytic cascade.
Plant serpins are potent inhibitors of mammalian chymotrypsin-like serine proteases in vitro, the best-studied example being barley serpin Zx (BSZx), which is able to inhibit trypsin and chymotrypsin as well as several blood coagulation factors. However, close relatives of chymotrypsin-like serine proteases are absent in plants. The RCL of several serpins from wheat grain and rye contain poly-Q repeat sequences similar to those present in the prolamin storage proteins of the endosperm. It has therefore been suggested that plant serpins may function to inhibit proteases from insects or microbes that would otherwise digest grain storage proteins. In support of this hypothesis, specific plant serpins have been identified in the phloem sap of pumpkin (CmPS-1) and cucumber plants. Although an inverse correlation between up-regulation of CmPS-1 expression and aphid survival was observed, in vitro feeding experiments revealed that recombinant CmPS-1 did not appear to affect insect survival.
Alternative roles and protease targets for plant serpins have been proposed. The Arabidopsis serpin, AtSerpin1 (At1g47710; ), mediates set-point control over programmed cell death by targeting the 'Responsive to Desiccation-21' (RD21) papain-like cysteine protease. AtSerpin1 also inhibits metacaspase-like proteases in vitro. Two other Arabidopsis serpins, AtSRP2 (At2g14540) and AtSRP3 (At1g64030) appear to be involved in responses to DNA damage.
target="_blank" rel="nofollow"> Frank Church laboratory at University of North Carolina at Chapel Hill
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