- Email: [email protected]
Molecular gymnastics: serpin structure, folding and misfolding
Molecular gymnastics: serpin structure, folding and misfolding James C Whisstock and Stephen P Bottomley The native state of serpins represents a long-lived intermediate or metastable structure on the serpin folding pathway. Upon interaction with a protease, the serpin trap is sprung and the molecule continues to fold into a more stable conformation. However, thermodynamic stability can also be achieved through alternative, unproductive folding pathways that result in the formation of inactive conformations. Our increasing understanding of the mechanism of protease inhibition and the dynamics of native serpin structures has begun to reveal how evolution has harnessed the actual process of protein folding (rather than the final folded outcome) to elegantly achieve function. The cost of using metastability for function, however, is an increased propensity for misfolding. Addresses Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, Clayton Campus, Melbourne 3800, Australia Corresponding author: Whisstock, James C ([email protected])
a 20–25 amino acid flexible loop (the reactive centre loop [RCL]) is positioned in an extended conformation above the body of the molecule (Figure 1a). The RCL functions as ‘bait’ for a target protease and the sequence of this region is the primary determinant of inhibitory specificity. Following initial protease docking (Figure 1b) and RCL cleavage (Figure 1c), the serpin scaffold undergoes an extensive conformational rearrangement whereby the RCL is incorporated into the body of the molecule as an additional central b-strand in the A b-sheet (Figure 1d) [6]. Conformational change also results in a dramatic increase in the overall stability of the serpin (native mammalian serpins typically exhibit a thermal melt of 40–60 8C in comparison to >100 8C for cleaved serpins [7,9]). Importantly, the serpin ‘trap’ is sprung before completion of the catalytic cycle of the protease and, as a result, the protease is ensnared at the acyl–enzyme intermediate stage and remains covalently attached to the serpin in the final serpin–enzyme complex (Figure 1d) [6]. Serpins are thus suicide inhibitors.
Current Opinion in Structural Biology 2006, 16:761–768 This review comes from a themed issue on Proteins Edited by Martino Bolognesi and Janet L Smith Available online 31st October 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.10.005
Introduction: an overview of the serpin inhibitory mechanism Serpins are the largest and most widely distributed family of protease inhibitors, bona fide inhibitory serpins having been identified in all major branches of life [1,2,3]. Around 90% of characterised serpins inhibit chymotrypsin-like serine proteases [4], although increasing numbers of cross-class serpin inhibitors of cysteine proteases have been identified ([5]; see also Update). In contrast to the small (10–15 kDa) and relatively rigid Kazal-type and Kunitz-like serine protease inhibitors and cystatin-like cysteine protease inhibitors, a typical serpin is large (35– 45 kDa) and relies on an extensive conformational change to achieve inhibitory function [6]. Since 1984, over eighty crystal structures of serpins have been determined, representing five different conformations. These data reveal that, rather than adopting the most stable conformation, serpins fold into a native, metastable state (Figure 1a) [7,8]. In this conformation, www.sciencedirect.com
On the face of it, serpins may seem like ‘Rube Goldberg’ machines, appearing to represent an unnecessarily complicated solution to the problem of protease inhibition! However, the serpin scaffold provides several important evolutionary advantages (summarized in Figures 1 and 2) over more standard ‘lock and key’-like protease inhibitors, including the ability to function as highly controllable sensors and inhibitors of proteolysis. Conversely, complexity renders the serpin scaffold vulnerable to nonproductive outcomes, most notably the formation of domain-swapped loop-sheet polymers [10] (Figure 3). A key advance in our understanding of serpin function and dysfunction was the recognition that the native conformation of serpins represented a metastable intermediate on the serpin folding pathway and that the formation of inactive serpin conformers, such as polymers, represented misfolding events [7,11]. Serpin-related diseases (serpinopathies) thus belong to a larger group of misfolding diseases that include poly-Q repeat disease, Alzheimer’s and prion diseases [12]. Here, we review the recent literature and highlight recent advances in our understanding of serpin mechanism, polymerisation and spontaneous conformational change. We show how a detailed investigation of serpin folding is providing a uniting link between our understanding of serpin mechanism and dysfunction.
Getting metastable and staying there The most stable conformation of the native serpin chain is the latent RCL-inserted form [13] (Figures 2 and 3). The native conformation of plasminogen activator inhibitor-1 Current Opinion in Structural Biology 2006, 16:761–768
762 Proteins
Figure 1
Inhibitory mechanism of serpins. (a) Native inhibitory antitrypsin (I; PDB code 1QLP [8]) contains three b-sheets and nine a-helices. The largest sheet (A) is in red and is labelled. The RCL is in blue at the top of the molecule. (b) Upon interaction with a protease (E; in pink), an initial docking or Michaelis complex is formed (E–I; PDB code 1OPH [48]). The residue primarily responsible for specificity, the P1 residue, is shown as blue spheres. (c) Cleavage of the RCL results in the incorporation of this region as a central, fourth strand (in blue) within the A b-sheet (E–I*; PDB code IEZX [6]). (d) The structure of the serpin–enzyme complex revealed a covalent linkage between the active site serine of the protease and the carbonyl oxygen of the P1 residue [6]. All cartoons made using PYMOL (Delano Scientific Ltd).
(PAI-1) spontaneously undergoes the transition to the latent form; this represents an elegant inbuilt mechanism for controlling inhibitory activity. However, because of a large energetic barrier, most inhibitory serpins do not readily attain this state and instead the folding pathway is prematurely interrupted at the native conformation (Figure 4) [14]. The potential for folding through to the latent conformation raises the question: how is the native state of inhibitory serpins attained and maintained? Or, vice versa, how does a folding serpin molecule avoid other thermodynamically more favourable conformations? Although these questions remain difficult to answer, recent folding studies are beginning to give clues as to how this is achieved. The culmination of several studies [15–22] suggests the following minimal pathway for serpin folding (Figure 4): U,I,N)L Where ‘U’ is the unfolded ensemble; ‘I’ represents a transiently stable, partially folded ensemble; ‘N’ represents the natively folded protein; and ‘L’ represents the latent conformation. Equilibrium folding studies of mesophilic serpins have revealed that the presence of I, populated at low denaCurrent Opinion in Structural Biology 2006, 16:761–768
turant concentrations, appears to be common to all serpins studied to date [15,16,20,21,23]. In addition, a more limited set of kinetic unfolding studies have identified a similar intermediate species, suggesting that there is a single intermediate ensemble through which all serpins pass to attain the native conformation [18,21,24]. Biophysical analysis by our laboratory and others suggests that this species contains approximately 80% of the native secondary structure, with partially formed A and C b-sheets, a well-formed B b-sheet and a non-native F helix [17,20,22,24,25,26]. Debate exists as to whether folding intermediates are productive or whether they take the protein ‘off’ the folding pathway, functioning as kinetic traps that hinder the folding process. Intermediates often slow the folding process because they contain non-native contacts that establish a kinetic barrier to the native state. In the case of serpins, non-native contacts may be productive and indeed obligatory in the attainment of the native state [14]. In particular, non-native interactions made by residues of the F-helix may play an important role in preventing misfolding, and it is suggested that the top of the F-helix may be inserted between strands 3A and 5A in the intermediate ensemble [27]. Such a conformation may be protective, as it would prevent self-insertion of the RCL and the transition to latency, as well as preventing the formation of loop-sheet polymers. As such, the existence of a stable intermediate on the serpin folding pathway appears essential to the attainment of a metastable native conformation. As the transition to the latent state represents the final step in serpin folding, the metastable native state may be termed a ‘folding intermediate’. Unfortunately, our understanding of the final N ) L step in serpin folding is limited. However, we can gain some insight into this pathway from our understanding of the dynamics of native serpins and serpin–enzyme complex formation.
Dynamics of the native serpin conformation: implications for the inhibitory mechanism and the transition to latency Both cleaved and latent serpins adopt RCL-inserted states that are structurally similar and display high thermal stabilities. Therefore, the transition from N ) L can be considered similar to the transition from N ) I*, where I* represents the cleaved serpin in the final serpin–enzyme complex. Thus, interaction with a target protease and RCL cleavage essentially lowers the kinetic barrier or offers an alternative energetic route to this more stable conformation. However, it is unclear precisely how RCL cleavage triggers conformational change. To begin to address this problem, it is important to consider the dynamics of the metastable state itself. Critically, it is becoming apparent that the concept of a native serpin as a ‘static’ metastable conformation, www.sciencedirect.com
Serpins Whisstock and Bottomley 763
Figure 2
Advantages of the serpin mechanism. Colour scheme is as used in Figure 1 unless otherwise stated. (a) The conformational mobility of serpins allows their activity to be modulated by specific cofactors. The structure of monomeric native antithrombin is shown (PDB code 1T1F; DJ Johnson et al., unpublished). The RCL of antithrombin is partially inserted into the top of the A b-sheet. The P1 residue (blue spheres) forms a salt bridge with Glu237 on the body of the serpin. This interaction partially occludes the P1 residue and explains why native antithrombin is a relatively poor inhibitor of thrombin and factor Xa [28]. (b) Upon interaction with a specific pentasaccharide sequence (green spheres) present in the cofactor heparin (yellow spheres), the RCL is expelled and the P1 residue is flipped into an exposed conformation, where it is more available to interact with the target proteases thrombin and factor Xa (PDB code 1TB6 [28,30]). It is suggested that an equilibrium between the native and RCL-expelled forms exists in vivo [28]. (c) The cofactor is also able to bind thrombin (pink) and accelerate inhibitor–protease interaction via a templating effect. (d) As a result of the requirement for RCL cleavage, serpins are ‘suicide’ one-use-only inhibitors and therefore represent an accurate method for controlling and (more importantly) removing proteases from a biological system (also depicted in Figure 1). Importantly, as a result of their conformational change, serpins act as molecular sensors of proteolysis that can signal the presence of proteases to the surrounding biological infrastructure. (e) Certain serpins, most notably PAI-1, contain in-built autocontrol mechanisms and spontaneously undergo the conformational change to an inactive monomeric latent conformation (PDB code 1DVN [13]). It is important to note that the formation of the latent form by many other serpins is associated with disease.
waiting for interaction with a protease, is an oversimplification. Recently determined structures of native serpins and Michaelis complexes with inactive proteases suggest that native serpins exist in a dynamic equilibrium or conformational ensembles in which the RCL flickers between a partially inserted and a fully expelled conformation (see www.sciencedirect.com
also Update). Thus, crystal structures of native serpins may ‘trap’ the most highly populated conformer [28,29,30] and events such as cofactor binding may function to shift the equilibrium towards the RCLexpelled form (Figure 2a,b) [28,30,31]. Supporting this idea, it has recently been demonstrated using hydrogen/deuterium exchange studies that much of the native serpin scaffold is substantially more flexible than Current Opinion in Structural Biology 2006, 16:761–768
764 Proteins
Figure 3
Disadvantages of the serpin mechanism. Colour scheme is as used in Figure 1. (a) Certain mutations, most often within the RCL itself, can interfere with the serpin mechanism and allow the protease to escape the serpin trap; this results in cleaved inactive serpin and free active protease. (b) More insidiously, serpins are vulnerable to mutations that result in misfolding and the formation of conformations that are no longer able to inhibit target proteases. In particular, many serpins are able to form inactive domain-swapped loop-sheet polymers (PDB code 1D5S; adapted from [49]), whereby the RCL of one serpin molecule is proposed to insert into the centre of the A b-sheet of another, forming a long-chain polymer. Polymerisation results in serpin deficiency and the failure to properly control proteolytic cascades. However, serpin polymers are also retained in the ER, where accumulation of misfolded material can eventually result in cell death and massive tissue damage. (c) Related to polymerisation, many inactivating mutations of serpins also result in spontaneous RCL insertion and inappropriate formation of the inactive latent conformation (illustrated using PDB structure 1DVN), again resulting in serpin deficiency. Several disease-linked mutations of human serpins have been identified that result in the formation of both polymeric and latent conformers [50].
previously anticipated [32]. Thus, interaction with a target protease followed by RCL cleavage may function to permit a rapid shift in the equilibrium towards a partially inserted conformation and, subsequently, towards full RCL insertion. It seems likely that protease distortion and inactivation must occur relatively rapidly during this process, so as to avoid hydrolysis of the acylenzyme intermediate and escape of active protease (Figure 3) [6,22,33].
Non-productive serpin folding: polymerisation and latency In 1992, the identification of the polymerogenic properties of the Z (Glu342 ! Lys) allele of antitrypsin opened the door to the study of serpin dysfunction through misfolding [10]. Since this discovery, more than forty variants of five different human serpins (antitrypsin, antichymotrypsin, antithrombin, C1 inhibitor and neuroserpin) have been identified that result in serpin-related misfolding diseases (serpinopathies) [3,34–36]. All these variants share several common features, including the ability to undergo spontaneous conformational change, Current Opinion in Structural Biology 2006, 16:761–768
decreased thermal stability, and the formation of polymeric forms that can be detected both in vivo and in vitro. At the conceptual level, it is suggested that the ability of the serpin scaffold to accept its own RCL renders it liable to accept the RCL of another molecule. Thus, serpin conformational change can be considered a race, whereby ‘self’ or cis RCL-insertion events must occur fast enough to block ‘foreign’ or trans RCL insertion. More recently, extensive biophysical studies have started to provide a detailed understanding of the molecular processes driving serpin polymerisation. Conformational change and polymerisation have been characterised most extensively for the serpins a1-antitrypsin, neuroserpin, a1-antichymotrypsin and PAI-1 [37–39,40,41–43]. Analysis of the spectroscopic changes during serpin polymerisation reveals the presence of a fast, concentration-independent phase, corresponding to the formation of a non-native species (M*). In common with other misfolding diseases, the formation of M* is the first step in polymerisation. It is suggested that M* occurs after the formation of the native state (N) www.sciencedirect.com
Serpins Whisstock and Bottomley 765
Figure 4
where ‘N’ represents the natively folded metastable monomeric form of the serpin; ‘M*’ represents the polymerogenic, conformationally altered monomeric serpin; ‘protodimer’ represents the dissociable dimeric species; and ‘P’ represents the polymeric species. a1-Antichymotrypsin polymerisation, however, is more complicated, as it is nucleation dependent [41,42]. The molecular details of this reaction are not known.
Schematic energy landscape illustrating the relationship between folding and conformation. As a result of the limited experimental data describing the kinetic and thermodynamic relationship between various serpin conformers, we present a highly simplified model, illustrating the folding of the serpin polypeptide chain based on the work described in this review. The surface ‘funnels’ the multitude of unfolded species through the folding intermediate ensemble (of unknown structure) to the unique but dynamic native structure. The blue spheres represent a highly simplified trajectory for the folding of the native molecule. From here, the molecule can continue to adopt more stable conformations, either by misfolding (red spheres) or by functional folding (green spheres). Although the latent conformation is shown as an example of serpin dysfunction, for certain serpins (e.g. PAI-1), formation of the latent state represents a proper functional outcome.
and is similar to, but distinct from, the intermediate state (I) that serpins must fold through en route to the native state. After formation of M*, the polymerisation processes demonstrate slower kinetic phases whose rates correlate positively with increasing protein concentration. These data are consistent with the formation of a dimeric species, followed by irreversible formation of oligomers via stabilization and elongation of the dimers: k1
k2
k3
N !M þ M !Protodimer!P www.sciencedirect.com
Structural elucidation of the serpin polymerogenic intermediate (M*) represents an important step in understanding serpin polymerisation and may prove essential for the rational design of therapeutics to block polymerisation. However, M* is formed transiently and is probably structurally heterogeneous. Despite these challenges, a growing body of biochemical evidence suggests that a primary effect of polymerogenic mutations such as Z-antitrypsin may be to shift the equilibrium between a native and partially inserted state (discussed earlier and shown in Figure 2) towards partial RCL insertion. Mahadeva et al. [44] show that an exogenous 6-mer peptide anneals to the lower portion of the A b-sheet in preference to the insertion of a full-length 12-mer peptide. These authors argue that 12-mer peptide insertion is blocked by the presence of RCL residues at the top of the A b-sheet. Additional support for partial insertion of the RCL in the polymerogenic intermediate comes from studies illustrating that the proximal hinge residues of the RCL in Z-antitrypsin have enhanced resistance to proteolytic attack [45]. Moreover, the annealing of short peptides to the upper part of the A b-sheet of antithrombin promotes polymerisation [46] and variants of the C1 inhibitor that promote partial insertion of the RCL spontaneously form multimers [47]. Together, these data strongly indicate that disruption of the top of the A-sheet by partial insertion of the RCL is important for the initiation of polymerisation. It is unclear, however, how serpins such as antithrombin and heparin cofactor II, which already have, or tend towards, a partially inserted conformation, avoid polymerisation. Presumably, these molecules have evolved strategies to prevent unwanted RCL insertion. Interestingly, it is worth noting that polymerogenic variants of RCL-inserted states of molecules such as antithrombin and antichymotrypsin readily adopt the latent, as well as the polymeric, form (Figure 3). Thus, it seems possible that, when serpins are pushed onto the pathway of inappropriate conformational change, latency and polymerisation compete, both pathways resulting in a final thermodynamically stable albeit functionally inactive outcome.
Conclusions Inhibitory serpins undergo a dramatic conformational rearrangement that is required for protease inhibition. However, serpins are markedly susceptible to mutations that result in the formation of inactive states. The analysis Current Opinion in Structural Biology 2006, 16:761–768
766 Proteins
of over eighty different X-ray crystal structures of serpins in five different conformational states, together with biophysical studies of serpin folding, has started to reveal a detailed picture of the molecular dynamics of conformational change. Crucially, the native structure of inhibitory serpins represents a long-lived intermediate or metastable state on the serpin folding pathway. During conformational rearrangement and protease inhibition, serpins switch from the metastable state to a stable conformation. Serpin ‘conformational change’ can therefore more accurately be considered as a continuation of the folding pathway that ultimately arrives at its thermodynamic minimum. Furthermore, thermodynamically stable, but functionally inactive, conformations, such as serpin polymers, are the result of misfolding events or the presence of unproductive branches of the serpin folding pathway.
Update
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
2.
Kang S, Barak Y, Lamed R, Bayer EA, Morrison M: The functional repertoire of prokaryote cellulosomes includes the serpin superfamily of serine proteinase inhibitors. Mol Microbiol 2006, 60:1344-1354. This work describes the presence of a bacterial serpin in the prokaryote cellulosome. The authors suggest that a possible role for bacterial serpins is to protect cellulosomes against proteolytic attack. 3.
Law RH, Zhang Q, McGowan S, Buckle AM, Silverman GA, Wong W, Rosado CJ, Langendorf CG, Pike RN, Bird PI et al.: An overview of the serpin superfamily. Genome Biol 2006, 7:216.
4.
Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, Irving JA, Lomas DA, Luke CJ, Moyer RW et al.: The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 2001, 276:33293-33296.
Function of a plant serpin
The precise role of serpins in plants has long been the subject of much debate and controversy. Vercammen et al. [51] have shown that serpin1 of Arabidopsis thaliana is an effective inhibitor of AtMC9, a type II (Arg/Lysspecific) metacaspase (a cysteine protease distantly related to animal caspases). Although the function of most metacaspases is unknown, a recent study revealed that one of these proteases (mcII-Pa) may play a role in programmed cell death in plants [52]. The work of Vercammen et al. [51] suggests a possible role for plant serpins in the regulation of metacaspase activity.
5.
McGowan S, Buckle AM, Irving JA, Ong PC, BashtannykPuhalovich TA, Kan WT, Henderson KN, Bulynko YA, Popova EY, Smith AI et al.: X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation. EMBO J 2006, 25:3144-3155. This work reveals that serpin loop-sheet polymers may play a functional role in chromatin condensation by nuclear serpins. Furthermore, it also reveals that intracellular serpins can adopt the partially inserted conformation. 6.
Huntington JA, Read RJ, Carrell RW: Structure of a serpinprotease complex shows inhibition by deformation. Nature 2000, 407:923-926.
7.
Carrell RW, Owen MC: Plakalbumin, alpha 1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis. Nature 1985, 317:730-732.
8.
Elliott PR, Lomas DA, Carrell RW, Abrahams JP: Inhibitory conformation of the reactive loop of alpha 1-antitrypsin. Nat Struct Biol 1996, 3:676-681.
9.
Dafforn TR, Pike RN, Bottomley SP: Physical characterization of serpin conformations. Methods 2004, 32:150-158.
Structure of thyroxine-binding globulin
In a recent study, Zhou et al. [53] determined the crystal structure of thyroxine-binding globulin (TGB) in complex with thyroxine. This is the first structure of a hormone-binding serpin and reveals that the ligand is wedged into a pocket formed by helices A and H, and strands 3–5 of the B-sheet. Interestingly, thyroxinebound TBG adopts a similar conformation to antithrombin, two residues of the RCL being partially inserted into the A-sheet. Significantly, the authors show how further insertion of the RCL from the partially inserted state to the d-conformation (in which four residues are inserted into the A-sheet) would result in thyroxine release. It is therefore suggested that a dynamic equilibrium between the partially inserted form and the d-conformation of TBG is linked to the binding and release of thyroxine. This elegant study further demonstrates the extraordinary flexibility and dynamics of the serpin scaffold.
Acknowledgements JCW is a National Health and Medical Research Council (NHMRC) senior research fellow and Monash University senior Logan fellow. SPB is an NHMRC R Douglas Wright fellow and a Monash University senior Logan fellow. We thank Mary Pearce, Ashley Buckle and Michelle Dunstone for critical reading of the manuscript, and the Australian Research Council and NHMRC for support. Current Opinion in Structural Biology 2006, 16:761–768
Irving JA, Pike RN, Lesk AM, Whisstock JC: Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 2000, 10:1841-1860.
10. Lomas DA, Evans DL, Finch JT, Carrell RW: The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature 1992, 357:605-607. 11. Berkenpas MB, Lawrence DA, Ginsburg D: Molecular evolution of plasminogen activator inhibitor-1 functional stability. EMBO J 1995, 14:2969-2977. 12. Chow MK, Lomas DA, Bottomley SP: Promiscuous beta-strand interactions and the conformational diseases. Curr Med Chem 2004, 11:491-499. 13. Mottonen J, Strand A, Symersky J, Sweet RM, Danley DE, Geoghegan KF, Gerard RD, Goldsmith EJ: Structural basis of latency in plasminogen activator inhibitor-1. Nature 1992, 355:270-273. 14. Cabrita LD, Bottomley SP: How do proteins avoid becoming too stable? Biophysical studies into metastable proteins. Eur Biophys J 2004, 33:83-88. 15. Villanueva GB, Allen N: Demonstration of a two-domain structure of antithrombin III during its denaturation in guanidinium chloride. J Biol Chem 1983, 258:11010-11013. 16. Herve M, Ghelis C: Conformational changes in intact and papain-modified alpha 1-proteinase inhibitor induced by guanidinium chloride. Eur J Biochem 1990, 191:653-658. www.sciencedirect.com
Serpins Whisstock and Bottomley 767
17. Powell LM, Pain RH: Effects of glycosylation on the folding and stability of human, recombinant and cleaved alpha 1antitrypsin. J Mol Biol 1992, 224:241-252. 18. Kim D, Yu MH: Folding pathway of human alpha 1-antitrypsin: characterization of an intermediate that is active but prone to aggregation. Biochem Biophys Res Commun 1996, 226:378-384. 19. Wang Z, Mottonen J, Goldsmith EJ: Kinetically controlled folding of the serpin plasminogen activator inhibitor 1. Biochemistry 1996, 35:16443-16448. 20. James EL, Whisstock JC, Gore MG, Bottomley SP: Probing the unfolding pathway of alpha1-antitrypsin. J Biol Chem 1999, 274:9482-9488. 21. Pearce MC, Rubin H, Bottomley SP: Conformational change and intermediates in the unfolding of alpha 1-antichymotrypsin. J Biol Chem 2000, 275:28513-28518.
32. Tsutsui Y, Liu L, Gershenson A, Wintrode PL: The conformational dynamics of a metastable serpin studied by hydrogen exchange and mass spectrometry. Biochemistry 2006, 45:6561-6569. This excellent study applies hydrogen/deuterium exchange and mass spectrometry to the serpin antitrypsin. This approach illustrates the dynamic nature of the metastable native state, and identifies some surprising regions of stability and instability. 33. Lawrence DA, Olson ST, Muhammad S, Day DE, Kvassman JO, Ginsburg D, Shore JD: Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into beta-sheet A. J Biol Chem 2000, 275:5839-5844. 34. Devlin GL, Bottomley SP: A protein family under ‘stress’ - serpin stability, folding and misfolding. Front Biosci 2005, 10:288-299. 35. Lomas DA, Carrell RW: Serpinopathies and the conformational dementias. Nat Rev Genet 2002, 3:759-768.
22. Tew DJ, Bottomley SP: Probing the equilibrium denaturation of the serpin alpha(1)-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state. J Mol Biol 2001, 313:1161-1169.
36. Stein PE, Carrell RW: What do dysfunctional serpins tell us about molecular mobility and disease? Nat Struct Biol 1995, 2:96-113.
23. Liu T, Pemberton PA, Robertson AD: Three-state unfolding and self-association of maspin, a tumor-suppressing serpin. J Biol Chem 1999, 274:29628-29632.
37. James EL, Bottomley SP: The mechanism of alpha 1-antitrypsin polymerization probed by fluorescence spectroscopy. Arch Biochem Biophys 1998, 356:296-300.
24. Cabrita LD, Dai W, Bottomley SP: Different conformational changes within the F-helix occur during serpin folding, polymerization, and proteinase inhibition. Biochemistry 2004, 43:9834-9839. A detailed energetic analysis of the role of the F-helix during serpin conformational change. A protein engineering approach shows that the F-helix undergoes strikingly different conformational changes during folding, misfolding and protease inhibition, suggesting potential control mechanisms for each of these.
38. Dafforn TR, Mahadeva R, Elliott PR, Sivasothy P, Lomas DA: A kinetic mechanism for the polymerization of alpha1antitrypsin. J Biol Chem 1999, 274:9548-9555.
25. Pearce MC, Cabrita LD, Rubin H, Gore MG, Bottomley SP: Identification of residual structure within denatured antichymotrypsin: implications for serpin folding and misfolding. Biochem Biophys Res Commun 2004, 324:729-735. 26. Cabrita LD, Whisstock JC, Bottomley SP: Probing the role of the F-helix in serpin stability through a single tryptophan substitution. Biochemistry 2002, 41:4575-4581. 27. Gooptu B, Hazes B, Chang WS, Dafforn TR, Carrell RW, Read RJ, Lomas DA: Inactive conformation of the serpin alpha(1)antichymotrypsin indicates two-stage insertion of the reactive loop: implications for inhibitory function and conformational disease. Proc Natl Acad Sci USA 2000, 97:67-72. 28. Li W, Johnson DJ, Esmon CT, Huntington JA: Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat Struct Mol Biol 2004, 11:857-862. The X-ray crystal structure of a ternary complex comprising antithrombin (serpin), thrombin (protease) and heparin (cofactor) reveals the structural basis of templating by heparin, as well as an exosite for thrombin on antithrombin. The structure also strongly suggests that an equilibrium exists in the ternary complex between a partially inserted and an RCLexpelled form. 29. Horvath AJ, Irving JA, Rossjohn J, Law RH, Bottomley SP, Quinsey NS, Pike RN, Coughlin PB, Whisstock JC: The murine orthologue of human antichymotrypsin: a structural paradigm for clade A3 serpins. J Biol Chem 2005, 280:43168-43178. 30. Johnson DJ, Li W, Adams TE, Huntington JA: Antithrombin S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation. EMBO J 2006, 25:2029-2037. The X-ray crystal structure of antithrombin (serpin), factor Xa (protease) and heparin (cofactor) reveals extensive exosite contacts between antithrombin and factor Xa. These data explain why heparin pentasaccharide alone is an effective activator of antithrombin compared with factor Xa. 31. Chuang YJ, Swanson R, Raja SM, Olson ST: Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin. J Biol Chem 2001, 276:14961-14971. www.sciencedirect.com
39. Zhou A, Faint R, Charlton P, Dafforn TR, Carrell RW, Lomas DA: Polymerization of plasminogen activator inhibitor-1. J Biol Chem 2001, 276:9115-9122. 40. Miranda E, Romisch K, Lomas DA: Mutants of neuroserpin that cause dementia accumulate as polymers within the endoplasmic reticulum. J Biol Chem 2004, 279:28283-28291. This study demonstrates that mutants of neuroserpin, which cause FENIB (familial encephalopathy with neuroserpin inclusion bodies), are retained as polymers within the endoplasmic reticulum (ER). The rate of accumulation was directly correlated with the severity of the clinical phenotype; intriguingly, however, the retained polymers were not toxic to the cell. 41. Crowther DC, Serpell LC, Dafforn TR, Gooptu B, Lomas DA: Nucleation of alpha(1)-antichymotrypsin polymerization. Biochemistry 2003, 42:2355-2363. 42. Devlin GL, Carver JA, Bottomley SP: The selective inhibition of serpin aggregation by the molecular chaperone, alphacrystallin, indicates a nucleation-dependent specificity. J Biol Chem 2003, 278:48644-48650. 43. Devlin GL, Chow MK, Howlett GJ, Bottomley SP: Acid denaturation of alpha1-antitrypsin: characterization of a novel mechanism of serpin polymerization. J Mol Biol 2002, 324:859-870. 44. Mahadeva R, Dafforn TR, Carrell RW, Lomas DA: 6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis. J Biol Chem 2002, 277:6771-6774. 45. Lomas DA, Evans DL, Stone SR, Chang WS, Carrell RW: Effect of the Z mutation on the physical and inhibitory properties of alpha 1-antitrypsin. Biochemistry 1993, 32:500-508. 46. Fitton HL, Pike RN, Carrell RW, Chang WS: Mechanisms of antithrombin polymerisation and heparin activation probed by the insertion of synthetic reactive loop peptides. Biol Chem 1997, 378:1059-1063. 47. Eldering E, Verpy E, Roem D, Meo T, Tosi M: COOH-terminal substitutions in the serpin c1 inhibitor that cause loop overinsertion and subsequent multimerization. J Biol Chem 1995, 270:2579-2587. 48. Dementiev A, Simonovic M, Volz K, Gettins PG: Canonical inhibitor-like interactions explain reactivity of alpha1proteinase inhibitor Pittsburgh and antithrombin with proteinases. J Biol Chem 2003, 278:37881-37887. Current Opinion in Structural Biology 2006, 16:761–768
768 Proteins
49. Dunstone MA, Dai W, Whisstock JC, Rossjohn J, Pike RN, Feil SC, Le Bonniec BF, Parker MW, Bottomley SP: Cleaved antitrypsin polymers at atomic resolution. Protein Sci 2000, 9:417-420. 50. Beauchamp NJ, Pike RN, Daly M, Butler L, Makris M, Dafforn TR, Zhou A, Fitton HL, Preston FE, Peake IR et al.: Antithrombins Wibble and Wobble (T85M/K): archetypal conformational diseases with in vivo latent-transition, thrombosis, and heparin activation. Blood 1998, 92:2696-2706. 51. Vercammen D, Belenghi B, van de Cotte B, Beunens T, Gavigan JA, De Rycke R, Brackenier A, Inze D, Harris JL, Van Breusegem F: Serpin1 of Arabidopsis thaliana is a suicide inhibitor for metacaspase 9. J Mol Biol 2006, in press. This paper provides strong evidence that the physiological target of A. thaliana serpin1 is the metacaspase AtMC9. Before publication of this work, the only serpin–caspase interaction characterised in vivo was the
Current Opinion in Structural Biology 2006, 16:761–768
interaction between the viral serpin crmA and the caspase ICE-1. This study provides evidence that serpins in plants may function in protecting against accidental release of pro-apoptotic proteases. 52. Bozhkov PV, Suarez MF, Filonova LH, Daniel G, Zamyatnin AA Jr, Rodriguez-Nieto S, Zhivotovsky B, Smertenko A: Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis. Proc Natl Acad Sci USA 2005, 102:14463-14468. 53. Zhou A, Wei Z, Read RJ, Carrell RW: Structural mechanism for the carriage and release of thyroxine in the blood. Proc Natl Acad Sci USA 2006, 103:13321-13326. This outstanding paper describes the structure of thyroxine-binding globulin. The work reveals how the conformation of the RCL directly influences the hormone-binding pocket that underlies the A-sheet. These data further elegantly demonstrate how serpin conformational change has been harnessed to reversibly regulate ligand binding and release.
www.sciencedirect.com
a 20–25 amino acid flexible loop (the reactive centre loop [RCL]) is positioned in an extended conformation above the body of the molecule (Figure 1a). The RCL functions as ‘bait’ for a target protease and the sequence of this region is the primary determinant of inhibitory specificity. Following initial protease docking (Figure 1b) and RCL cleavage (Figure 1c), the serpin scaffold undergoes an extensive conformational rearrangement whereby the RCL is incorporated into the body of the molecule as an additional central b-strand in the A b-sheet (Figure 1d) [6]. Conformational change also results in a dramatic increase in the overall stability of the serpin (native mammalian serpins typically exhibit a thermal melt of 40–60 8C in comparison to >100 8C for cleaved serpins [7,9]). Importantly, the serpin ‘trap’ is sprung before completion of the catalytic cycle of the protease and, as a result, the protease is ensnared at the acyl–enzyme intermediate stage and remains covalently attached to the serpin in the final serpin–enzyme complex (Figure 1d) [6]. Serpins are thus suicide inhibitors.
Current Opinion in Structural Biology 2006, 16:761–768 This review comes from a themed issue on Proteins Edited by Martino Bolognesi and Janet L Smith Available online 31st October 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.10.005
Introduction: an overview of the serpin inhibitory mechanism Serpins are the largest and most widely distributed family of protease inhibitors, bona fide inhibitory serpins having been identified in all major branches of life [1,2,3]. Around 90% of characterised serpins inhibit chymotrypsin-like serine proteases [4], although increasing numbers of cross-class serpin inhibitors of cysteine proteases have been identified ([5]; see also Update). In contrast to the small (10–15 kDa) and relatively rigid Kazal-type and Kunitz-like serine protease inhibitors and cystatin-like cysteine protease inhibitors, a typical serpin is large (35– 45 kDa) and relies on an extensive conformational change to achieve inhibitory function [6]. Since 1984, over eighty crystal structures of serpins have been determined, representing five different conformations. These data reveal that, rather than adopting the most stable conformation, serpins fold into a native, metastable state (Figure 1a) [7,8]. In this conformation, www.sciencedirect.com
On the face of it, serpins may seem like ‘Rube Goldberg’ machines, appearing to represent an unnecessarily complicated solution to the problem of protease inhibition! However, the serpin scaffold provides several important evolutionary advantages (summarized in Figures 1 and 2) over more standard ‘lock and key’-like protease inhibitors, including the ability to function as highly controllable sensors and inhibitors of proteolysis. Conversely, complexity renders the serpin scaffold vulnerable to nonproductive outcomes, most notably the formation of domain-swapped loop-sheet polymers [10] (Figure 3). A key advance in our understanding of serpin function and dysfunction was the recognition that the native conformation of serpins represented a metastable intermediate on the serpin folding pathway and that the formation of inactive serpin conformers, such as polymers, represented misfolding events [7,11]. Serpin-related diseases (serpinopathies) thus belong to a larger group of misfolding diseases that include poly-Q repeat disease, Alzheimer’s and prion diseases [12]. Here, we review the recent literature and highlight recent advances in our understanding of serpin mechanism, polymerisation and spontaneous conformational change. We show how a detailed investigation of serpin folding is providing a uniting link between our understanding of serpin mechanism and dysfunction.
Getting metastable and staying there The most stable conformation of the native serpin chain is the latent RCL-inserted form [13] (Figures 2 and 3). The native conformation of plasminogen activator inhibitor-1 Current Opinion in Structural Biology 2006, 16:761–768
762 Proteins
Figure 1
Inhibitory mechanism of serpins. (a) Native inhibitory antitrypsin (I; PDB code 1QLP [8]) contains three b-sheets and nine a-helices. The largest sheet (A) is in red and is labelled. The RCL is in blue at the top of the molecule. (b) Upon interaction with a protease (E; in pink), an initial docking or Michaelis complex is formed (E–I; PDB code 1OPH [48]). The residue primarily responsible for specificity, the P1 residue, is shown as blue spheres. (c) Cleavage of the RCL results in the incorporation of this region as a central, fourth strand (in blue) within the A b-sheet (E–I*; PDB code IEZX [6]). (d) The structure of the serpin–enzyme complex revealed a covalent linkage between the active site serine of the protease and the carbonyl oxygen of the P1 residue [6]. All cartoons made using PYMOL (Delano Scientific Ltd).
(PAI-1) spontaneously undergoes the transition to the latent form; this represents an elegant inbuilt mechanism for controlling inhibitory activity. However, because of a large energetic barrier, most inhibitory serpins do not readily attain this state and instead the folding pathway is prematurely interrupted at the native conformation (Figure 4) [14]. The potential for folding through to the latent conformation raises the question: how is the native state of inhibitory serpins attained and maintained? Or, vice versa, how does a folding serpin molecule avoid other thermodynamically more favourable conformations? Although these questions remain difficult to answer, recent folding studies are beginning to give clues as to how this is achieved. The culmination of several studies [15–22] suggests the following minimal pathway for serpin folding (Figure 4): U,I,N)L Where ‘U’ is the unfolded ensemble; ‘I’ represents a transiently stable, partially folded ensemble; ‘N’ represents the natively folded protein; and ‘L’ represents the latent conformation. Equilibrium folding studies of mesophilic serpins have revealed that the presence of I, populated at low denaCurrent Opinion in Structural Biology 2006, 16:761–768
turant concentrations, appears to be common to all serpins studied to date [15,16,20,21,23]. In addition, a more limited set of kinetic unfolding studies have identified a similar intermediate species, suggesting that there is a single intermediate ensemble through which all serpins pass to attain the native conformation [18,21,24]. Biophysical analysis by our laboratory and others suggests that this species contains approximately 80% of the native secondary structure, with partially formed A and C b-sheets, a well-formed B b-sheet and a non-native F helix [17,20,22,24,25,26]. Debate exists as to whether folding intermediates are productive or whether they take the protein ‘off’ the folding pathway, functioning as kinetic traps that hinder the folding process. Intermediates often slow the folding process because they contain non-native contacts that establish a kinetic barrier to the native state. In the case of serpins, non-native contacts may be productive and indeed obligatory in the attainment of the native state [14]. In particular, non-native interactions made by residues of the F-helix may play an important role in preventing misfolding, and it is suggested that the top of the F-helix may be inserted between strands 3A and 5A in the intermediate ensemble [27]. Such a conformation may be protective, as it would prevent self-insertion of the RCL and the transition to latency, as well as preventing the formation of loop-sheet polymers. As such, the existence of a stable intermediate on the serpin folding pathway appears essential to the attainment of a metastable native conformation. As the transition to the latent state represents the final step in serpin folding, the metastable native state may be termed a ‘folding intermediate’. Unfortunately, our understanding of the final N ) L step in serpin folding is limited. However, we can gain some insight into this pathway from our understanding of the dynamics of native serpins and serpin–enzyme complex formation.
Dynamics of the native serpin conformation: implications for the inhibitory mechanism and the transition to latency Both cleaved and latent serpins adopt RCL-inserted states that are structurally similar and display high thermal stabilities. Therefore, the transition from N ) L can be considered similar to the transition from N ) I*, where I* represents the cleaved serpin in the final serpin–enzyme complex. Thus, interaction with a target protease and RCL cleavage essentially lowers the kinetic barrier or offers an alternative energetic route to this more stable conformation. However, it is unclear precisely how RCL cleavage triggers conformational change. To begin to address this problem, it is important to consider the dynamics of the metastable state itself. Critically, it is becoming apparent that the concept of a native serpin as a ‘static’ metastable conformation, www.sciencedirect.com
Serpins Whisstock and Bottomley 763
Figure 2
Advantages of the serpin mechanism. Colour scheme is as used in Figure 1 unless otherwise stated. (a) The conformational mobility of serpins allows their activity to be modulated by specific cofactors. The structure of monomeric native antithrombin is shown (PDB code 1T1F; DJ Johnson et al., unpublished). The RCL of antithrombin is partially inserted into the top of the A b-sheet. The P1 residue (blue spheres) forms a salt bridge with Glu237 on the body of the serpin. This interaction partially occludes the P1 residue and explains why native antithrombin is a relatively poor inhibitor of thrombin and factor Xa [28]. (b) Upon interaction with a specific pentasaccharide sequence (green spheres) present in the cofactor heparin (yellow spheres), the RCL is expelled and the P1 residue is flipped into an exposed conformation, where it is more available to interact with the target proteases thrombin and factor Xa (PDB code 1TB6 [28,30]). It is suggested that an equilibrium between the native and RCL-expelled forms exists in vivo [28]. (c) The cofactor is also able to bind thrombin (pink) and accelerate inhibitor–protease interaction via a templating effect. (d) As a result of the requirement for RCL cleavage, serpins are ‘suicide’ one-use-only inhibitors and therefore represent an accurate method for controlling and (more importantly) removing proteases from a biological system (also depicted in Figure 1). Importantly, as a result of their conformational change, serpins act as molecular sensors of proteolysis that can signal the presence of proteases to the surrounding biological infrastructure. (e) Certain serpins, most notably PAI-1, contain in-built autocontrol mechanisms and spontaneously undergo the conformational change to an inactive monomeric latent conformation (PDB code 1DVN [13]). It is important to note that the formation of the latent form by many other serpins is associated with disease.
waiting for interaction with a protease, is an oversimplification. Recently determined structures of native serpins and Michaelis complexes with inactive proteases suggest that native serpins exist in a dynamic equilibrium or conformational ensembles in which the RCL flickers between a partially inserted and a fully expelled conformation (see www.sciencedirect.com
also Update). Thus, crystal structures of native serpins may ‘trap’ the most highly populated conformer [28,29,30] and events such as cofactor binding may function to shift the equilibrium towards the RCLexpelled form (Figure 2a,b) [28,30,31]. Supporting this idea, it has recently been demonstrated using hydrogen/deuterium exchange studies that much of the native serpin scaffold is substantially more flexible than Current Opinion in Structural Biology 2006, 16:761–768
764 Proteins
Figure 3
Disadvantages of the serpin mechanism. Colour scheme is as used in Figure 1. (a) Certain mutations, most often within the RCL itself, can interfere with the serpin mechanism and allow the protease to escape the serpin trap; this results in cleaved inactive serpin and free active protease. (b) More insidiously, serpins are vulnerable to mutations that result in misfolding and the formation of conformations that are no longer able to inhibit target proteases. In particular, many serpins are able to form inactive domain-swapped loop-sheet polymers (PDB code 1D5S; adapted from [49]), whereby the RCL of one serpin molecule is proposed to insert into the centre of the A b-sheet of another, forming a long-chain polymer. Polymerisation results in serpin deficiency and the failure to properly control proteolytic cascades. However, serpin polymers are also retained in the ER, where accumulation of misfolded material can eventually result in cell death and massive tissue damage. (c) Related to polymerisation, many inactivating mutations of serpins also result in spontaneous RCL insertion and inappropriate formation of the inactive latent conformation (illustrated using PDB structure 1DVN), again resulting in serpin deficiency. Several disease-linked mutations of human serpins have been identified that result in the formation of both polymeric and latent conformers [50].
previously anticipated [32]. Thus, interaction with a target protease followed by RCL cleavage may function to permit a rapid shift in the equilibrium towards a partially inserted conformation and, subsequently, towards full RCL insertion. It seems likely that protease distortion and inactivation must occur relatively rapidly during this process, so as to avoid hydrolysis of the acylenzyme intermediate and escape of active protease (Figure 3) [6,22,33].
Non-productive serpin folding: polymerisation and latency In 1992, the identification of the polymerogenic properties of the Z (Glu342 ! Lys) allele of antitrypsin opened the door to the study of serpin dysfunction through misfolding [10]. Since this discovery, more than forty variants of five different human serpins (antitrypsin, antichymotrypsin, antithrombin, C1 inhibitor and neuroserpin) have been identified that result in serpin-related misfolding diseases (serpinopathies) [3,34–36]. All these variants share several common features, including the ability to undergo spontaneous conformational change, Current Opinion in Structural Biology 2006, 16:761–768
decreased thermal stability, and the formation of polymeric forms that can be detected both in vivo and in vitro. At the conceptual level, it is suggested that the ability of the serpin scaffold to accept its own RCL renders it liable to accept the RCL of another molecule. Thus, serpin conformational change can be considered a race, whereby ‘self’ or cis RCL-insertion events must occur fast enough to block ‘foreign’ or trans RCL insertion. More recently, extensive biophysical studies have started to provide a detailed understanding of the molecular processes driving serpin polymerisation. Conformational change and polymerisation have been characterised most extensively for the serpins a1-antitrypsin, neuroserpin, a1-antichymotrypsin and PAI-1 [37–39,40,41–43]. Analysis of the spectroscopic changes during serpin polymerisation reveals the presence of a fast, concentration-independent phase, corresponding to the formation of a non-native species (M*). In common with other misfolding diseases, the formation of M* is the first step in polymerisation. It is suggested that M* occurs after the formation of the native state (N) www.sciencedirect.com
Serpins Whisstock and Bottomley 765
Figure 4
where ‘N’ represents the natively folded metastable monomeric form of the serpin; ‘M*’ represents the polymerogenic, conformationally altered monomeric serpin; ‘protodimer’ represents the dissociable dimeric species; and ‘P’ represents the polymeric species. a1-Antichymotrypsin polymerisation, however, is more complicated, as it is nucleation dependent [41,42]. The molecular details of this reaction are not known.
Schematic energy landscape illustrating the relationship between folding and conformation. As a result of the limited experimental data describing the kinetic and thermodynamic relationship between various serpin conformers, we present a highly simplified model, illustrating the folding of the serpin polypeptide chain based on the work described in this review. The surface ‘funnels’ the multitude of unfolded species through the folding intermediate ensemble (of unknown structure) to the unique but dynamic native structure. The blue spheres represent a highly simplified trajectory for the folding of the native molecule. From here, the molecule can continue to adopt more stable conformations, either by misfolding (red spheres) or by functional folding (green spheres). Although the latent conformation is shown as an example of serpin dysfunction, for certain serpins (e.g. PAI-1), formation of the latent state represents a proper functional outcome.
and is similar to, but distinct from, the intermediate state (I) that serpins must fold through en route to the native state. After formation of M*, the polymerisation processes demonstrate slower kinetic phases whose rates correlate positively with increasing protein concentration. These data are consistent with the formation of a dimeric species, followed by irreversible formation of oligomers via stabilization and elongation of the dimers: k1
k2
k3
N !M þ M !Protodimer!P www.sciencedirect.com
Structural elucidation of the serpin polymerogenic intermediate (M*) represents an important step in understanding serpin polymerisation and may prove essential for the rational design of therapeutics to block polymerisation. However, M* is formed transiently and is probably structurally heterogeneous. Despite these challenges, a growing body of biochemical evidence suggests that a primary effect of polymerogenic mutations such as Z-antitrypsin may be to shift the equilibrium between a native and partially inserted state (discussed earlier and shown in Figure 2) towards partial RCL insertion. Mahadeva et al. [44] show that an exogenous 6-mer peptide anneals to the lower portion of the A b-sheet in preference to the insertion of a full-length 12-mer peptide. These authors argue that 12-mer peptide insertion is blocked by the presence of RCL residues at the top of the A b-sheet. Additional support for partial insertion of the RCL in the polymerogenic intermediate comes from studies illustrating that the proximal hinge residues of the RCL in Z-antitrypsin have enhanced resistance to proteolytic attack [45]. Moreover, the annealing of short peptides to the upper part of the A b-sheet of antithrombin promotes polymerisation [46] and variants of the C1 inhibitor that promote partial insertion of the RCL spontaneously form multimers [47]. Together, these data strongly indicate that disruption of the top of the A-sheet by partial insertion of the RCL is important for the initiation of polymerisation. It is unclear, however, how serpins such as antithrombin and heparin cofactor II, which already have, or tend towards, a partially inserted conformation, avoid polymerisation. Presumably, these molecules have evolved strategies to prevent unwanted RCL insertion. Interestingly, it is worth noting that polymerogenic variants of RCL-inserted states of molecules such as antithrombin and antichymotrypsin readily adopt the latent, as well as the polymeric, form (Figure 3). Thus, it seems possible that, when serpins are pushed onto the pathway of inappropriate conformational change, latency and polymerisation compete, both pathways resulting in a final thermodynamically stable albeit functionally inactive outcome.
Conclusions Inhibitory serpins undergo a dramatic conformational rearrangement that is required for protease inhibition. However, serpins are markedly susceptible to mutations that result in the formation of inactive states. The analysis Current Opinion in Structural Biology 2006, 16:761–768
766 Proteins
of over eighty different X-ray crystal structures of serpins in five different conformational states, together with biophysical studies of serpin folding, has started to reveal a detailed picture of the molecular dynamics of conformational change. Crucially, the native structure of inhibitory serpins represents a long-lived intermediate or metastable state on the serpin folding pathway. During conformational rearrangement and protease inhibition, serpins switch from the metastable state to a stable conformation. Serpin ‘conformational change’ can therefore more accurately be considered as a continuation of the folding pathway that ultimately arrives at its thermodynamic minimum. Furthermore, thermodynamically stable, but functionally inactive, conformations, such as serpin polymers, are the result of misfolding events or the presence of unproductive branches of the serpin folding pathway.
Update
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
2.
Kang S, Barak Y, Lamed R, Bayer EA, Morrison M: The functional repertoire of prokaryote cellulosomes includes the serpin superfamily of serine proteinase inhibitors. Mol Microbiol 2006, 60:1344-1354. This work describes the presence of a bacterial serpin in the prokaryote cellulosome. The authors suggest that a possible role for bacterial serpins is to protect cellulosomes against proteolytic attack. 3.
Law RH, Zhang Q, McGowan S, Buckle AM, Silverman GA, Wong W, Rosado CJ, Langendorf CG, Pike RN, Bird PI et al.: An overview of the serpin superfamily. Genome Biol 2006, 7:216.
4.
Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, Irving JA, Lomas DA, Luke CJ, Moyer RW et al.: The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 2001, 276:33293-33296.
Function of a plant serpin
The precise role of serpins in plants has long been the subject of much debate and controversy. Vercammen et al. [51] have shown that serpin1 of Arabidopsis thaliana is an effective inhibitor of AtMC9, a type II (Arg/Lysspecific) metacaspase (a cysteine protease distantly related to animal caspases). Although the function of most metacaspases is unknown, a recent study revealed that one of these proteases (mcII-Pa) may play a role in programmed cell death in plants [52]. The work of Vercammen et al. [51] suggests a possible role for plant serpins in the regulation of metacaspase activity.
5.
McGowan S, Buckle AM, Irving JA, Ong PC, BashtannykPuhalovich TA, Kan WT, Henderson KN, Bulynko YA, Popova EY, Smith AI et al.: X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation. EMBO J 2006, 25:3144-3155. This work reveals that serpin loop-sheet polymers may play a functional role in chromatin condensation by nuclear serpins. Furthermore, it also reveals that intracellular serpins can adopt the partially inserted conformation. 6.
Huntington JA, Read RJ, Carrell RW: Structure of a serpinprotease complex shows inhibition by deformation. Nature 2000, 407:923-926.
7.
Carrell RW, Owen MC: Plakalbumin, alpha 1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis. Nature 1985, 317:730-732.
8.
Elliott PR, Lomas DA, Carrell RW, Abrahams JP: Inhibitory conformation of the reactive loop of alpha 1-antitrypsin. Nat Struct Biol 1996, 3:676-681.
9.
Dafforn TR, Pike RN, Bottomley SP: Physical characterization of serpin conformations. Methods 2004, 32:150-158.
Structure of thyroxine-binding globulin
In a recent study, Zhou et al. [53] determined the crystal structure of thyroxine-binding globulin (TGB) in complex with thyroxine. This is the first structure of a hormone-binding serpin and reveals that the ligand is wedged into a pocket formed by helices A and H, and strands 3–5 of the B-sheet. Interestingly, thyroxinebound TBG adopts a similar conformation to antithrombin, two residues of the RCL being partially inserted into the A-sheet. Significantly, the authors show how further insertion of the RCL from the partially inserted state to the d-conformation (in which four residues are inserted into the A-sheet) would result in thyroxine release. It is therefore suggested that a dynamic equilibrium between the partially inserted form and the d-conformation of TBG is linked to the binding and release of thyroxine. This elegant study further demonstrates the extraordinary flexibility and dynamics of the serpin scaffold.
Acknowledgements JCW is a National Health and Medical Research Council (NHMRC) senior research fellow and Monash University senior Logan fellow. SPB is an NHMRC R Douglas Wright fellow and a Monash University senior Logan fellow. We thank Mary Pearce, Ashley Buckle and Michelle Dunstone for critical reading of the manuscript, and the Australian Research Council and NHMRC for support. Current Opinion in Structural Biology 2006, 16:761–768
Irving JA, Pike RN, Lesk AM, Whisstock JC: Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 2000, 10:1841-1860.
10. Lomas DA, Evans DL, Finch JT, Carrell RW: The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature 1992, 357:605-607. 11. Berkenpas MB, Lawrence DA, Ginsburg D: Molecular evolution of plasminogen activator inhibitor-1 functional stability. EMBO J 1995, 14:2969-2977. 12. Chow MK, Lomas DA, Bottomley SP: Promiscuous beta-strand interactions and the conformational diseases. Curr Med Chem 2004, 11:491-499. 13. Mottonen J, Strand A, Symersky J, Sweet RM, Danley DE, Geoghegan KF, Gerard RD, Goldsmith EJ: Structural basis of latency in plasminogen activator inhibitor-1. Nature 1992, 355:270-273. 14. Cabrita LD, Bottomley SP: How do proteins avoid becoming too stable? Biophysical studies into metastable proteins. Eur Biophys J 2004, 33:83-88. 15. Villanueva GB, Allen N: Demonstration of a two-domain structure of antithrombin III during its denaturation in guanidinium chloride. J Biol Chem 1983, 258:11010-11013. 16. Herve M, Ghelis C: Conformational changes in intact and papain-modified alpha 1-proteinase inhibitor induced by guanidinium chloride. Eur J Biochem 1990, 191:653-658. www.sciencedirect.com
Serpins Whisstock and Bottomley 767
17. Powell LM, Pain RH: Effects of glycosylation on the folding and stability of human, recombinant and cleaved alpha 1antitrypsin. J Mol Biol 1992, 224:241-252. 18. Kim D, Yu MH: Folding pathway of human alpha 1-antitrypsin: characterization of an intermediate that is active but prone to aggregation. Biochem Biophys Res Commun 1996, 226:378-384. 19. Wang Z, Mottonen J, Goldsmith EJ: Kinetically controlled folding of the serpin plasminogen activator inhibitor 1. Biochemistry 1996, 35:16443-16448. 20. James EL, Whisstock JC, Gore MG, Bottomley SP: Probing the unfolding pathway of alpha1-antitrypsin. J Biol Chem 1999, 274:9482-9488. 21. Pearce MC, Rubin H, Bottomley SP: Conformational change and intermediates in the unfolding of alpha 1-antichymotrypsin. J Biol Chem 2000, 275:28513-28518.
32. Tsutsui Y, Liu L, Gershenson A, Wintrode PL: The conformational dynamics of a metastable serpin studied by hydrogen exchange and mass spectrometry. Biochemistry 2006, 45:6561-6569. This excellent study applies hydrogen/deuterium exchange and mass spectrometry to the serpin antitrypsin. This approach illustrates the dynamic nature of the metastable native state, and identifies some surprising regions of stability and instability. 33. Lawrence DA, Olson ST, Muhammad S, Day DE, Kvassman JO, Ginsburg D, Shore JD: Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into beta-sheet A. J Biol Chem 2000, 275:5839-5844. 34. Devlin GL, Bottomley SP: A protein family under ‘stress’ - serpin stability, folding and misfolding. Front Biosci 2005, 10:288-299. 35. Lomas DA, Carrell RW: Serpinopathies and the conformational dementias. Nat Rev Genet 2002, 3:759-768.
22. Tew DJ, Bottomley SP: Probing the equilibrium denaturation of the serpin alpha(1)-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state. J Mol Biol 2001, 313:1161-1169.
36. Stein PE, Carrell RW: What do dysfunctional serpins tell us about molecular mobility and disease? Nat Struct Biol 1995, 2:96-113.
23. Liu T, Pemberton PA, Robertson AD: Three-state unfolding and self-association of maspin, a tumor-suppressing serpin. J Biol Chem 1999, 274:29628-29632.
37. James EL, Bottomley SP: The mechanism of alpha 1-antitrypsin polymerization probed by fluorescence spectroscopy. Arch Biochem Biophys 1998, 356:296-300.
24. Cabrita LD, Dai W, Bottomley SP: Different conformational changes within the F-helix occur during serpin folding, polymerization, and proteinase inhibition. Biochemistry 2004, 43:9834-9839. A detailed energetic analysis of the role of the F-helix during serpin conformational change. A protein engineering approach shows that the F-helix undergoes strikingly different conformational changes during folding, misfolding and protease inhibition, suggesting potential control mechanisms for each of these.
38. Dafforn TR, Mahadeva R, Elliott PR, Sivasothy P, Lomas DA: A kinetic mechanism for the polymerization of alpha1antitrypsin. J Biol Chem 1999, 274:9548-9555.
25. Pearce MC, Cabrita LD, Rubin H, Gore MG, Bottomley SP: Identification of residual structure within denatured antichymotrypsin: implications for serpin folding and misfolding. Biochem Biophys Res Commun 2004, 324:729-735. 26. Cabrita LD, Whisstock JC, Bottomley SP: Probing the role of the F-helix in serpin stability through a single tryptophan substitution. Biochemistry 2002, 41:4575-4581. 27. Gooptu B, Hazes B, Chang WS, Dafforn TR, Carrell RW, Read RJ, Lomas DA: Inactive conformation of the serpin alpha(1)antichymotrypsin indicates two-stage insertion of the reactive loop: implications for inhibitory function and conformational disease. Proc Natl Acad Sci USA 2000, 97:67-72. 28. Li W, Johnson DJ, Esmon CT, Huntington JA: Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat Struct Mol Biol 2004, 11:857-862. The X-ray crystal structure of a ternary complex comprising antithrombin (serpin), thrombin (protease) and heparin (cofactor) reveals the structural basis of templating by heparin, as well as an exosite for thrombin on antithrombin. The structure also strongly suggests that an equilibrium exists in the ternary complex between a partially inserted and an RCLexpelled form. 29. Horvath AJ, Irving JA, Rossjohn J, Law RH, Bottomley SP, Quinsey NS, Pike RN, Coughlin PB, Whisstock JC: The murine orthologue of human antichymotrypsin: a structural paradigm for clade A3 serpins. J Biol Chem 2005, 280:43168-43178. 30. Johnson DJ, Li W, Adams TE, Huntington JA: Antithrombin S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation. EMBO J 2006, 25:2029-2037. The X-ray crystal structure of antithrombin (serpin), factor Xa (protease) and heparin (cofactor) reveals extensive exosite contacts between antithrombin and factor Xa. These data explain why heparin pentasaccharide alone is an effective activator of antithrombin compared with factor Xa. 31. Chuang YJ, Swanson R, Raja SM, Olson ST: Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin. J Biol Chem 2001, 276:14961-14971. www.sciencedirect.com
39. Zhou A, Faint R, Charlton P, Dafforn TR, Carrell RW, Lomas DA: Polymerization of plasminogen activator inhibitor-1. J Biol Chem 2001, 276:9115-9122. 40. Miranda E, Romisch K, Lomas DA: Mutants of neuroserpin that cause dementia accumulate as polymers within the endoplasmic reticulum. J Biol Chem 2004, 279:28283-28291. This study demonstrates that mutants of neuroserpin, which cause FENIB (familial encephalopathy with neuroserpin inclusion bodies), are retained as polymers within the endoplasmic reticulum (ER). The rate of accumulation was directly correlated with the severity of the clinical phenotype; intriguingly, however, the retained polymers were not toxic to the cell. 41. Crowther DC, Serpell LC, Dafforn TR, Gooptu B, Lomas DA: Nucleation of alpha(1)-antichymotrypsin polymerization. Biochemistry 2003, 42:2355-2363. 42. Devlin GL, Carver JA, Bottomley SP: The selective inhibition of serpin aggregation by the molecular chaperone, alphacrystallin, indicates a nucleation-dependent specificity. J Biol Chem 2003, 278:48644-48650. 43. Devlin GL, Chow MK, Howlett GJ, Bottomley SP: Acid denaturation of alpha1-antitrypsin: characterization of a novel mechanism of serpin polymerization. J Mol Biol 2002, 324:859-870. 44. Mahadeva R, Dafforn TR, Carrell RW, Lomas DA: 6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis. J Biol Chem 2002, 277:6771-6774. 45. Lomas DA, Evans DL, Stone SR, Chang WS, Carrell RW: Effect of the Z mutation on the physical and inhibitory properties of alpha 1-antitrypsin. Biochemistry 1993, 32:500-508. 46. Fitton HL, Pike RN, Carrell RW, Chang WS: Mechanisms of antithrombin polymerisation and heparin activation probed by the insertion of synthetic reactive loop peptides. Biol Chem 1997, 378:1059-1063. 47. Eldering E, Verpy E, Roem D, Meo T, Tosi M: COOH-terminal substitutions in the serpin c1 inhibitor that cause loop overinsertion and subsequent multimerization. J Biol Chem 1995, 270:2579-2587. 48. Dementiev A, Simonovic M, Volz K, Gettins PG: Canonical inhibitor-like interactions explain reactivity of alpha1proteinase inhibitor Pittsburgh and antithrombin with proteinases. J Biol Chem 2003, 278:37881-37887. Current Opinion in Structural Biology 2006, 16:761–768
768 Proteins
49. Dunstone MA, Dai W, Whisstock JC, Rossjohn J, Pike RN, Feil SC, Le Bonniec BF, Parker MW, Bottomley SP: Cleaved antitrypsin polymers at atomic resolution. Protein Sci 2000, 9:417-420. 50. Beauchamp NJ, Pike RN, Daly M, Butler L, Makris M, Dafforn TR, Zhou A, Fitton HL, Preston FE, Peake IR et al.: Antithrombins Wibble and Wobble (T85M/K): archetypal conformational diseases with in vivo latent-transition, thrombosis, and heparin activation. Blood 1998, 92:2696-2706. 51. Vercammen D, Belenghi B, van de Cotte B, Beunens T, Gavigan JA, De Rycke R, Brackenier A, Inze D, Harris JL, Van Breusegem F: Serpin1 of Arabidopsis thaliana is a suicide inhibitor for metacaspase 9. J Mol Biol 2006, in press. This paper provides strong evidence that the physiological target of A. thaliana serpin1 is the metacaspase AtMC9. Before publication of this work, the only serpin–caspase interaction characterised in vivo was the
Current Opinion in Structural Biology 2006, 16:761–768
interaction between the viral serpin crmA and the caspase ICE-1. This study provides evidence that serpins in plants may function in protecting against accidental release of pro-apoptotic proteases. 52. Bozhkov PV, Suarez MF, Filonova LH, Daniel G, Zamyatnin AA Jr, Rodriguez-Nieto S, Zhivotovsky B, Smertenko A: Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis. Proc Natl Acad Sci USA 2005, 102:14463-14468. 53. Zhou A, Wei Z, Read RJ, Carrell RW: Structural mechanism for the carriage and release of thyroxine in the blood. Proc Natl Acad Sci USA 2006, 103:13321-13326. This outstanding paper describes the structure of thyroxine-binding globulin. The work reveals how the conformation of the RCL directly influences the hormone-binding pocket that underlies the A-sheet. These data further elegantly demonstrate how serpin conformational change has been harnessed to reversibly regulate ligand binding and release.
www.sciencedirect.com