transition). the serpin (white) first binds a protease (grey) with the exposed reactive centre loop (blue). when this loop is cleaved by the protease, it rapidly inserts into the a-sheet (light blue), deforming and inhibiting the protease. (pdb: 1k9o, 1ezx​)serine and cysteine proteases operate by a two-step catalytic mechanism. first, the substrate (blue) is attacked by the cysteine or serine of the catalytic triad (red) to form an acyl-enzyme intermediate. for typical substrates, the intermediate is resolved by hydrolysis by water. however, when the reactive centre loop (rcl) of a serpin is attacked, the conformational change (blue arrow) pulls the catalytic triad out of position, preventing it from completing catalysis. (based on pdb: 1k9o, 1ezx​) allosteric activation some serpins are activated by cofactors. the serpin antithrombin has an rcl (blue) where the p1 arginine (blue sticks) points inwards, preventing protease binding. binding of heparin (green sticks) causes the p1 arginine residue to flip to an exposed position. the target protease (grey) then binds to both the exposed p1 arginine as well as the heparin. the serpin then activates and heparin is released. (pdb: 1tb6, 2ant, 1tb6, 1ezx​) the conformational mobility of serpins provides a key advantage over static lock-and-key protease inhibitors. in particular, the function of inhibitory serpins can be regulated by allosteric interactions with specific cofactors. the x-ray crystal structures of antithrombin, heparin cofactor ii, ment and murine antichymotrypsin reveal that these serpins adopt a conformation wherein the first two amino acids of the rcl are inserted into the top of the a β-sheet. the partially inserted conformation is important because co-factors are able to conformationally switch certain partially inserted serpins into a fully expelled form. this conformational rearrangement makes the serpin a more effective inhibitor. 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 anti-clotting drug. latent conformation some serpins can spontaneously convert to an inactive latent state. the serpin pai-1 remains in the active conformation when bound to vitronectin (green). however, in the absence of vitronectin, pai-1 can change to the inactive latent state. the uncleaved rcl (blue; disordered regions as dashed lines) inserts into the a-sheet, pulling a β-strand off the c-sheet (yellow). (pdb: 1oc0, 1dvm, 1lj5​) certain serpins spontaneously undergo the s to r transition without having been cleaved by a protease, to form a conformation termed the latent state. latent serpins are unable to interact with proteases and so are no longer protease inhibitors. the conformational change to latency is not exactly the same as the s to r transition of a cleaved serpin. since the rcl is still intact, the first strand of the c-sheet has to peel off to allow full rcl insertion. 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 tengpin, 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. conformational change in non-inhibitory functions certain non-inhibitory serpins also use the serpin conformational change as part of their function. for example, the native (s) form of thyroxine-binding globulin has high affinity for thyroxine, whereas the cleaved (r) form has low affinity. similarly, transcortin has higher affinity for cortisol when in its native (s) state, than its cleaved (r) state. thus, in these serpins, rcl cleavage and the s to r transition has been commandeered to allow for ligand release, rather than protease inhibition. in some serpins, the s to r transition can activate cell signalling 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. degradation when a serpin inhibits a target protease, it forms a permanent complex, which needs to be disposed of. for extracellular serpins, the final serpin-enzyme complexes are rapidly cleared from circulation. one mechanism by which this occurs in mammals is via the low-density lipoprotein receptor-related protein (lrp), which binds to inhibitory complexes made by antithrombin, pa1-1, and neuroserpin, causing cellular uptake. similarly, the drosophila serpin, necrotic, is degraded in the lysosome after being trafficked into the cell by the lipophorin receptor-1 (homologous to the mammalian ldl receptor family). disease and serpinopathies serpins are involved in a wide array of physiological functions, and so mutations in genes encoding them can cause a range of diseases. mutations that change the activity, specificity or aggregation properties of serpins all affect how they function. the majority of serpin-related diseases are the result of serpin polymerisation into aggregates, though several other types of disease-linked mutations also occur. the disorder alpha-1 antitrypsin deficiency is one of the most common hereditary diseases. inactivity or absence the inactive δ-conformation of the disease-linked antichymotrypsin mutant (l55p). four residues of the rcl (blue; disordered region as dashed line) are inserted into the top of the a-sheet. part of the f α-helix (yellow) has unwound and fills the bottom half of the a-sheet. (pdb: 1qmn​) since the stressed serpin fold is high-energy, mutations can cause them to incorrectly change into their lower-energy conformations (e.g. relaxed or latent) before they have correctly performed their inhibitory role. 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. gene knockouts, particularly in mice, are used experimentally to determine the normal functions of serpins by the effect of their absence. specificity change in some rare cases, a single amino acid change in a serpin's rcl alters its specificity to target the wrong protease. for example, the antitrypsin-pittsburgh mutation (m358r) causes the α1-antitrypsin serpin to inhibit thrombin, causing a bleeding disorder. polymerisation and aggregation serpin polymerisation by domain swappinga domain-swapped serpin dimer. (pdb: 2znh​)a domain-swapped serpin trimer. each monomer's rcl is inserted into its own structure (shown in red of the green monomer). (pdb: 3t1p​) the majority of serpin diseases are due to protein aggregation and are termed "serpinopathies". serpins are vulnerable to disease-causing mutations that promote formation of misfolded polymers due to their inherently unstable structures. well-characterised serpinopathies include α1-antitrypsin deficiency (alpha-1), which may cause familial emphysema, and sometimes liver cirrhosis, certain familial forms of thrombosis related to antithrombin deficiency, types 1 and 2 hereditary angioedema (hae) related to deficiency of c1-inhibitor, and familial encephalopathy with neuroserpin inclusion bodies (fenib; a rare type of dementia caused by neuroserpin polymerisation). 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 proteopathies (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 liver cells, 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 domain swapping 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 dimer and 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. therapeutic strategies several therapeutic approaches are in use or under investigation to treat the most common serpinopathy: antitrypsin deficiency. antitrypsin augmentation therapy is approved for severe antitrypsin deficiency-related emphysema. in this therapy, antitrypsin is purified from the plasma of blood donors and administered intravenously (first marketed as prolastin). to treat severe antitrypsin deficiency-related disease, lung and liver transplantation has proven effective. in animal models, gene targeting in induced pluripotent stem cells has been successfully used to correct an antitrypsin polymerisation defect and to restore the ability of the mammalian liver to secrete active antitrypsin. small molecules have also been developed that block antitrypsin polymerisation in vitro. evolution serpins are the most widely distributed and largest superfamily of protease inhibitors. they were initially believed to be restricted to eukaryote organisms, but have since been found in bacteria, archaea and some viruses. it remains unclear whether prokaryote genes are the descendants of an ancestral prokaryotic serpin or the product of horizontal gene transfer from eukaryotes. most intracellular serpins belong to a single phylogenetic clade, whether they come from plants or animals, indicating that the intracellular and extracellular serpins may have diverged before the plants and animals. exceptions include the intracellular heat shock serpin hsp47, which is a chaperone essential for proper folding of collagen, and cycles between the cis-golgi and the endoplasmic