serpin serpin serpins are a superfamily of proteins 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 proteases (serine protease inhibitors). 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. serpin (serine protease inhibitor)a serpin (white) with its 'reactive centre loop' (blue) bound to a protease (grey). once the protease attempts catalysis it will be irreversibly inhibited. (pdb: 1k9o​)identifierssymbolserpin, serpin (root symbol of family)pfampf00079interproipr000215prositepdoc00256scop21hle / scope / supfamcddcd00172available protein structures:pfam structures / ecod pdbrcsb pdb; pdbe; pdbjpdbsumstructure summarypdb1m37a:1-378 1hleb:349-379 1jrra:1-415 1by7a:1-415 1ovaa:1-385 1uhga:1-385 1jtib:1-385 1attb:77-433 1nq9l:76-461 1oyhi:76-461 1e03l:76-461 1e05i:76-461 1br8l:76-461 1r1ll:76-461 1lk6l:76-461 1antl:76-461 2behl:76-461 1dzhl:76-461 1atha:78-461 1tb6i:76-461 2anti:76-461p 1dzgi:76-461 1azxl:76-461 1jvqi:76-461 1sr5a:76-461 1e04i:76-461 1xqga:1-375 1xu8b:1-375 1wz9b:1-375 1xqja:1-375 1c8oa:1-300 1m93a:1-55 1f0ca:1-305 1k9oi:18-392 1sek :18-369 1atu :45-415 1ezxb:383-415 8apia:43-382 1qmba:49-376 1iz2a:43-415 1oo8a:43-415 1d5sb:378-415 7apia:44-382 1qlpa:43-415 1opha:43-415 1kct :44-415 2d26a:43-382 9apib:383-415 1psi :47-415 1hp7a:43-415 3caaa:50-383 1qmna:43-420 4caab:390-420 2acha:47-383 1as4a:48-383 1yxab:42-417 1lq8f:376-406 2paib:374-406 1paib:374-406 1jmoa:119-496 1jmja:119-496 1oc0a:25-402 1dvna:25-402 1b3kd:25-402 1dvmd:25-402 1a7ca:25-402 1c5ga:25-402 1db2b:26-402 9paia:25-402 1lj5a:25-402 1m6qa:138-498 1jjod:101-361 1imva:49-415 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 protein misfolding and the formation of inactive long-chain polymers. 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 proteolytic cascades, some proteins with a serpin structure are not enzyme inhibitors, but instead perform diverse functions such as storage (as in egg white—ovalbumin), transport as in hormone carriage proteins (thyroxine-binding globulin, cortisol-binding globulin) 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. history protease inhibitory activity in blood plasma was first reported in the late 1800s, but it was not until the 1950s that the serpins antithrombin and alpha 1-antitrypsin were isolated, with the subsequent recognition of their close family homology in 1979. that they belonged to a new protein family became apparent on their further alignment with the non-inhibitory egg-white protein ovalbumin, to give what was initially called the alpha1-antitrypsin-antithrombin iii-ovalbumin superfamily of serine proteinase inhibitors, but was subsequently succinctly renamed as the serpins. the initial characterisation of the new family centred on alpha1-antitrypsin, a serpin present in high concentration in blood plasma, the common genetic disorder of which was shown to cause a predisposition to the lung disease emphysema and to liver cirrhosis. the identification of the s and z mutations responsible for the genetic deficiency and the subsequent sequence alignments of alpha1-antitrypsin and antithrombin in 1982 led to the recognition of the close homologies of the active sites of the two proteins, centred on a methionine in alpha1-antitrypsin as an inhibitor of tissue elastase and on arginine in antithrombin as an inhibitor of thrombin. 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 viruses. 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. activity a protease (grey) bound to a serpin reactive centre loop (rcl, blue). when the protease's catalytic triad (red) cleaves the rcl, it becomes trapped in an inactive conformation. (pdb: 1k9o​) most serpins are protease inhibitors, targeting extracellular, chymotrypsin-like serine proteases. these proteases possess a nucleophilic serine residue in a catalytic triad in their active site. examples include thrombin, trypsin, and human neutrophil elastase. serpins act as irreversible, suicide inhibitors by trapping an intermediate of the protease's catalytic mechanism. 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. biological function and localization protease inhibition approximately two-thirds of human serpins perform extracellular roles, inhibiting proteases in the bloodstream in order to modulate their activities. for example, extracellular serpins regulate the proteolytic cascades central to blood clotting (antithrombin), the inflammatory and immune responses (antitrypsin, antichymotrypsin, and c1-inhibitor) and tissue remodelling (pai-1). by inhibiting signalling cascade proteases, they can also affect development. the table of human serpins (below) provides examples of the range of functions performed by human serpin, as well as some of the diseases that result from serpin deficiency. 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 cell death pathways. some viruses use serpins to disrupt protease functions in their host. the cowpox viral serpin crma (cytokine response modifier a) is used in order to avoid inflammatory and apoptotic 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 metacaspases and a papain-like cysteine protease. non-inhibitory roles non-inhibitory extracellular serpins also perform a wide array of important roles. thyroxine-binding globulin and transcortin transport the hormones thyroxine and cortisol, respectively. the non-inhibitory serpin ovalbumin is the most abundant protein in egg white. its exact function is unknown, but it is thought to be a storage protein for the developing foetus. heat shock serpin 47 is a chaperone, essential for proper folding of collagen. it acts by stabilising collagen's triple helix whilst it is being processed in the endoplasmic reticulum. some serpins are both protease inhibitors and perform additional roles. for example, the nuclear cysteine protease inhibitor ment, in birds also acts as a chromatin remodelling molecule in a bird's red blood cells. structure the native state of serpins is an equilibrium between a fully stressed state (left) and a partially relaxed state (right). the five-stranded a-sheet (light blue) contains two functionally important regions for the serpin's mechanism, the breach and the shutter. the reactive centre loop (rcl, blue) exists in a dynamic equilibrium between the fully exposed conformation (left) and a conformation where it is partially inserted into the breach of the a-sheet (right).(pdb: 1qlp, 1yxa​) all serpins share a common structure (or fold), despite their varied functions. all typically have three β-sheets (named a, b and c) and eight or nine α-helices (named ha–hi). the most significant regions to serpin function are the a-sheet and the reactive centre loop (rcl). the a-sheet includes two β-strands that are in a parallel orientation with a region between them called the 'shutter', and upper region called the 'breach'. the rcl forms the initial interaction with the target protease in inhibitory molecules. structures have been solved showing the rcl either fully exposed or partially inserted into the a-sheet, and serpins are thought to be in dynamic equilibrium between these two states. the rcl also only makes temporary interactions with the rest of the structure, and is therefore highly flexible and exposed to the solvent. 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. conformational change and inhibitory mechanism inhibitory serpins do not inhibit their target proteases by the typical competitive (lock-and-key) mechanism used by most small protease inhibitors (e.g. kunitz-type inhibitors). instead, serpins use an unusual conformational change, which disrupts the structure of the protease and prevents it from completing catalysis. the conformational change involves the rcl moving to the opposite end of the protein and inserting into β-sheet a, forming an extra antiparallel β-strand. this converts the serpin from a stressed state, to a lower-energy relaxed state (s to r transition). serine and cysteine proteases catalyse peptide bond cleavage by a two-step process. initially, the catalytic residue of the active site triad performs a nucleophilic 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 hydrolysed 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 kinetic rate 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 inhibitory mechanism of serpins involves a large conformational change (s to r