Aeropin from the Extremophile Pyrobaculum aerophilum Bypasses the Serpin Misfolding Trap

Serpins are metastable proteinase inhibitors. Serpin metastability drives both a large conformational change that is utilized during proteinase inhibition and confers an inherent structural flexibility that renders serpins susceptible to aggregation under certain conditions. These include point mutations (the basis of a number of important human genetic diseases), small changes in pH, and an increase in temperature. Many studies of serpins from mesophilic organisms have highlighted an inverse relationship: mutations that confer a marked increase in serpin stability compromise inhibitory activity. Here we present the first biophysical characterization of a metastable serpin from a hyperthermophilic organism. Aeropin, from the archaeon Pyrobaculum aerophilum, is both highly stable and an efficient proteinase inhibitor. We also demonstrate that because of high kinetic barriers, aeropin does not readily form the partially unfolded precursor to serpin aggregation. We conclude that stability and activity are not mutually exclusive properties in the context of the serpin fold, and propose that the increased stability of aeropin is caused by an unfolding pathway that minimizes the formation of an aggregation-prone intermediate ensemble, thereby enabling aeropin to bypass the misfolding fate observed with other serpins. Serpins are metastable proteinase inhibitors. Serpin metastability drives both a large conformational change that is utilized during proteinase inhibition and confers an inherent structural flexibility that renders serpins susceptible to aggregation under certain conditions. These include point mutations (the basis of a number of important human genetic diseases), small changes in pH, and an increase in temperature. Many studies of serpins from mesophilic organisms have highlighted an inverse relationship: mutations that confer a marked increase in serpin stability compromise inhibitory activity. Here we present the first biophysical characterization of a metastable serpin from a hyperthermophilic organism. Aeropin, from the archaeon Pyrobaculum aerophilum, is both highly stable and an efficient proteinase inhibitor. We also demonstrate that because of high kinetic barriers, aeropin does not readily form the partially unfolded precursor to serpin aggregation. We conclude that stability and activity are not mutually exclusive properties in the context of the serpin fold, and propose that the increased stability of aeropin is caused by an unfolding pathway that minimizes the formation of an aggregation-prone intermediate ensemble, thereby enabling aeropin to bypass the misfolding fate observed with other serpins. Members of the serine proteinase inhibitor (serpin) 6The abbreviations used are:serpinserine proteinase inhibitorRCLreactive center loopGdnHClguanidine hydrochlorideNTAnitrilotriacetic acidα1-ATarchetypal serpin antitrypsin 6The abbreviations used are:serpinserine proteinase inhibitorRCLreactive center loopGdnHClguanidine hydrochlorideNTAnitrilotriacetic acidα1-ATarchetypal serpin antitrypsin superfamily are predominantly proteinase inhibitors whose native conformation is metastable (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (828) Google Scholar, 2Cabrita L.D. Bottomley S.P. Eur. Biophys. J. 2004; 33: 83-88Crossref PubMed Scopus (35) Google Scholar, 3Whisstock J.C. Bottomley S.P. Curr. Opin. Struct. Biol. 2006; 16: 761-768Crossref PubMed Scopus (118) Google Scholar). They, therefore, represent an exception to the Anfinsen rule that all proteins fold to their most energetically preferred state (4Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5022) Google Scholar). Other metastable proteins include influenza hemagglutinin (5Bullough P.A. Hughson F.M. Skehel J.J. Wiley D.C. Nature. 1994; 371: 37-43Crossref PubMed Scopus (1365) Google Scholar) and α-lytic protease (6Baker D. Sohl J.L. Agard D.A. Nature. 1992; 356: 263-265Crossref PubMed Scopus (283) Google Scholar). All these proteins use their metastability as a source of stored potential energy that is expended to perform their biological function. serine proteinase inhibitor reactive center loop guanidine hydrochloride nitrilotriacetic acid archetypal serpin antitrypsin serine proteinase inhibitor reactive center loop guanidine hydrochloride nitrilotriacetic acid archetypal serpin antitrypsin For serpins, the metastable native state possesses an intrinsic structural flexibility, which permits a rapid conformational change that is required for proteinase inhibition. The propensity to undergo conformational change is supported by a highly conserved tertiary architecture, which consists of three β-sheets (A to C) surrounded by 9 α-helices (hA–hI) and a solvent-exposed reactive center loop (RCL), which determines inhibitory specificity (Fig. 1). During proteinase inhibition, the RCL is cleaved by the proteinase and becomes incorporated as a middle strand of the A-sheet, a process referred to as the stressed → relaxed (S to R) transition. As a result, the proteinase is translocated to the opposite pole of the serpin, and the two are trapped in a highly stable, covalent serpin-enzyme complex (7Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (932) Google Scholar). The serpin thus surrenders its metastability in favor of adopting a more stable conformation that complements proteinase inhibition. The energetic basis of this inhibitory mechanism is that incorporation of the RCL as a strand into the A-sheet is thermodynamically favorable (8Bruch M. Weiss V. Engel J. J. Biol. Chem. 1988; 263: 16626-16630Abstract Full Text PDF PubMed Google Scholar). However, serpins can also adopt another thermodynamically favorable state by inserting their RCL into an adjacent serpin molecule. Propagation of the resulting loop-sheet linkages results in the formation of long-chain A-sheet polymers (9Mast A.E. Enghild J.J. Salvesen G. Biochemistry. 1992; 31: 2720-2728Crossref PubMed Scopus (185) Google Scholar, 10Bottomley S.P. Hopkins P.C. Whisstock J.C. Biochem. Biophys. Res. Commun. 1998; 251: 1-5Crossref PubMed Scopus (24) Google Scholar, 11Dunstone M.A. Dai W. Whisstock J.C. Rossjohn J. Pike R.N. Feil S.C. Le Bonniec B.F. Parker M.W. Bottomley S.P. Protein Sci. 2000; 9: 417-420Crossref PubMed Scopus (81) Google Scholar, 12Huntington J.A. Pannu N.S. Hazes B. Read R.J. Lomas D.A. Carrell R.W. J. Mol. Biol. 1999; 293: 449-455Crossref PubMed Scopus (117) Google Scholar). Loop A-sheet polymers occur when the serpin native state is perturbed by mutation or small changes in pH and temperature. Such perturbation results in the partial unfolding of the serpin and the formation of a nonnative ensemble of structures, which then readily self-associate (13James E.L. Bottomley S.P. Arch. Biochem. Biophys. 1998; 356: 296-300Crossref PubMed Scopus (74) Google Scholar, 14Cabrita L.D. Dai W. Bottomley S.P. Biochemistry. 2004; 43: 9834-9839Crossref PubMed Scopus (55) Google Scholar, 15Purkayastha P. Klemke J.W. Lavender S. Oyola R. Cooperman B.S. Gai F. Biochemistry. 2005; 44: 2642-2649Crossref PubMed Scopus (33) Google Scholar). It has been shown that by increasing the stability of this intermediate ensemble or the rate of its formation, the likelihood of polymerization occurring is also significantly increased (16Yu M.H. Lee K.N. Kim J. Nat. Struct. Biol. 1995; 2: 363-367Crossref PubMed Scopus (139) Google Scholar). Physiologically, this is manifested in a range of loss-of-function diseases such as emphysema, liver cirrhosis, thrombosis, and dementia (17Lomas D.A. Carrell R.W. Nat. Rev. Genet. 2002; 3: 759-768Crossref PubMed Scopus (192) Google Scholar). An understanding of the determinants of metastability in serpins thus has relevance to important human pathologies. Extensive biochemical and biophysical studies have demonstrated that residues important to maintaining and controlling metastability are distributed throughout the molecule (18Seo E.J. Im H. Maeng J.S. Kim K.E. Yu M.H. J. Biol. Chem. 2000; 275: 16904-16909Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 19Lee C. Park S.H. Lee M.Y. Yu M.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7727-7731Crossref PubMed Scopus (96) Google Scholar). The introduction of mutations that destabilize the native state of a serpin increase its propensity to polymerize (20Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (389) Google Scholar, 21Gilis D. McLennan H.R. Dehouck Y. Cabrita L.D. Rooman M. Bottomley S.P. J. Mol. Biol. 2003; 325: 581-589Crossref PubMed Scopus (24) Google Scholar). Stabilizing mutations produce more diverse effects: some only moderately enhance stability and retain serpin function (19Lee C. Park S.H. Lee M.Y. Yu M.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7727-7731Crossref PubMed Scopus (96) Google Scholar, 21Gilis D. McLennan H.R. Dehouck Y. Cabrita L.D. Rooman M. Bottomley S.P. J. Mol. Biol. 2003; 325: 581-589Crossref PubMed Scopus (24) Google Scholar), while others yield increased stability at the expense of inhibition, presumably because the flexibility that is required for inhibition is no longer present (22Im H. Ryu M.J. Yu M.H. Protein Eng. Des. Sel. 2004; 17: 325-331Crossref PubMed Scopus (12) Google Scholar). These studies, which have been restricted to serpins from mesophilic organisms, highlight a serpin metastability paradigm that reflects an exquisite balance between structural stability and flexibility to achieve biological function. This structure/function compromise seemingly precludes the possibility of an active serpin with a greatly increased native state stability. However, this position is at odds with the presence of serpin sequences in the genomes of several moderate to highly thermophilic bacteria and archaea (23Irving J.A. Steenbakkers P.J. Lesk A.M. Op den Camp H.J. Pike R.N. Whisstock J.C. Mol. Biol. Evol. 2002; 19: 1881-1890Crossref PubMed Scopus (105) Google Scholar, 24Irving J.A. Cabrita L.D. Rossjohn J. Pike R.N. Bottomley S.P. Whisstock J.C. Structure (Camb.). 2003; 11: 387-397Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 25Fulton K.F. Buckle A.M. Cabrita L.D. Irving J.A. Butcher R.E. Smith I. Reeve S. Lesk A.M. Bottomley S.P. Rossjohn J. Whisstock J.C. J. Biol. Chem. 2005; 280: 8435-8442Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 26Zhang Q. Buckle A.M. Law R.H. Pearce M.C. Cabrita L.D. Lloyd G.J. Irving J.A. Smith A.I. Ruzyla K. Rossjohn J. Bottomley S.P. Whisstock J.C. EMBO Rep. 2007; 8: 658-663Crossref PubMed Scopus (46) Google Scholar). This article reports the first characterization of a serpin from a hyperthermophilic organism: the crenarchaeon, Pyrobaculum aerophilum, which thrives in deep thermal vents at temperatures exceeding 100 °C (23Irving J.A. Steenbakkers P.J. Lesk A.M. Op den Camp H.J. Pike R.N. Whisstock J.C. Mol. Biol. Evol. 2002; 19: 1881-1890Crossref PubMed Scopus (105) Google Scholar, 27Volkl P. Huber R. Drobner E. Rachel R. Burggraf S. Trincone A. Stetter K.O. Appl. Environ. Microbiol. 1993; 59: 2918-2926Crossref PubMed Google Scholar, 28Fitz-Gibbon S.T. Ladner H. Kim U.J. Stetter K.O. Simon M.I. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 984-989Crossref PubMed Scopus (196) Google Scholar). We demonstrate that this serpin, aeropin, possesses a remarkable resistance to chemical and thermal denaturation with respect to all other characterized serpins, while paradoxically remaining an effective proteinase inhibitor. More intriguingly, the data reveal a potential mechanism by which aeropin and other hyperthermophilic proteins are able to avoid aggregation. Cloning, Expression, and Purification—Genomic DNA from P. The and used to the aeropin and The into the to the The presence of the mutations with DNA The sequences used to the the mutation is and and and and For and the aeropin and the into of aeropin and the at an of and for at The both and It that the of to at to the a that has been for other proteins from P. C. S. J. Mol. Biol. 2000; PubMed Scopus Google Scholar, G. J. D. Biochemistry. PubMed Scopus Google Scholar). The most as by and stored at aeropin as the at 100 °C for to J.A. Cabrita L.D. Rossjohn J. Pike R.N. Bottomley S.P. Whisstock J.C. Structure (Camb.). 2003; 11: 387-397Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). kinetic in pH as Le Bonniec B.F. Hopkins P.C. K. Biochemistry. PubMed Scopus Google Scholar). of guanidine hydrochloride in pH and the PubMed Scopus Google Scholar). unfolding and as E.L. Whisstock J.C. Bottomley S.P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). (13James E.L. Bottomley S.P. Arch. Biochem. Biophys. 1998; 356: 296-300Crossref PubMed Scopus (74) Google Scholar). The at a of in pH for and between and 100 of aeropin at a of in The then a in pH a aeropin into an E. and the and to with its by not of aeropin with other serpins (Fig. that possesses most of the highly conserved residues of the serpin superfamily J.A. Pike R.N. Lesk A.M. Whisstock J.C. Res. 2000; PubMed Scopus Google Scholar), that to the serpin as shown in the in most the an also from the of M. Q. H.R. D. Salvesen Structure Des. 2000; 8: Full Text Full Text PDF Scopus Google Scholar), a significantly two The also that aeropin is to a proteinase inhibitor to the presence of an inhibitory (23Irving J.A. Steenbakkers P.J. Lesk A.M. Op den Camp H.J. Pike R.N. Whisstock J.C. Mol. Biol. Evol. 2002; 19: 1881-1890Crossref PubMed Scopus (105) Google Scholar) (Fig. The of the inhibitory specificity of a serpin is a residues to the P.C. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar), as the I. A. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). However, the proteinase by aeropin is not and the aeropin residues not propose a aeropin to a inhibitor with a of inhibition of of inhibitor required to molecule of proteinase at °C (Fig. and 1). under that the proteinase and inhibitor in a covalent inhibitory complex (Fig. the rate at °C to 1). these data that aeropin is a highly efficient proteinase that the serpin mechanism of inhibition, and aeropin is of the → and stability of aeropin and its not not in a thermal stability of aeropin by changes in during an increase in temperature. Aeropin, in no change in the range (Fig. which that its The thermal in the presence of which shown in to the native conformation of The presence of in an thermal unfolding from the with a of °C (Fig. and 1). It is to that under the the archetypal serpin antitrypsin used as a for throughout this is unfolded E.L. Whisstock J.C. Bottomley S.P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). These data demonstrate that aeropin possesses a with respect to all other inhibitory serpins to of serpin metastability is the to a more stable state of the aeropin the thermal stability of the form of aeropin As can in in the presence of cleaved aeropin does not at temperatures in of that RCL to an increase in stability. The of aeropin also (Fig. with the thermal aeropin °C which a of aeropin from the Such a presumably reflects aggregation and not polymerization as no with serpin polymerization This has been with other serpins Whisstock J. Hopkins P.C. Lesk A.M. Carrell R.W. Protein Sci. PubMed Scopus Google Scholar). to in the high stability of aeropin a pH by at pH and the of denaturation for both aeropin and to stable to changes in pH and not an unfolding the pH to a pH and unfolded at pH G.J. Bottomley S.P. J. Mol. Biol. 2002; PubMed Scopus Google of pH aeropin in a studies of aeropin both and intrinsic also in with other serpins aeropin not to active As a result, a not the the unfolding used as the of stability. change in the at which point a with a of and unfolding observed in (Fig. The unfolding to reveal the with a highly intermediate ensemble, that is of all other serpins to J.A. Cabrita L.D. Rossjohn J. Pike R.N. Bottomley S.P. Whisstock J.C. Structure (Camb.). 2003; 11: 387-397Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, E.L. Whisstock J.C. Bottomley S.P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.H. J. Mol. Biol. 1992; PubMed Scopus Google Scholar, M.C. H. Bottomley S.P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (33) Google Scholar, P.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. Eur. J. Biochem. PubMed Scopus Google Scholar). also used to As observed with the unfolding of aeropin a native to (Fig. which a highly to the unfolded state a of the and unfolding a unfolding the that are not that aeropin a with an unfolding The presence of an unfolding intermediate has been characterized in several serpins by their to the E.L. Whisstock J.C. Bottomley S.P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M.C. H. Bottomley S.P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (33) Google Scholar). the intermediate ensemble by aeropin such a we aeropin in the presence of and the resulting However, no change in the not that the intermediate ensemble possesses to by has been that at high and to polymerize D.A. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar). aeropin to aggregation during the unfolding aeropin in increasing and and a at observed (Fig. a of both unfolded and and also a small The of the with the of in the studies to produce an intermediate in aeropin with an at studies that serpins at and thus is that no aeropin present during unfolding not of to of partial unfolding of the native serpin state is the first in polymerization (13James E.L. Bottomley S.P. Arch. Biochem. Biophys. 1998; 356: 296-300Crossref PubMed Scopus (74) Google Scholar, R. P. Lomas D.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), we the unfolding rate of aeropin is a of the unfolding of aeropin in It is that aeropin a of and a kinetic the presence of two kinetic kinetic unfolding for aeropin a range of and the two of unfolding by as PubMed Scopus Google Scholar). The → rate to while the at These to that aggregation not occurring during the unfolding the of the in the of data have shown that aeropin has both high stability and inhibitory activity. a structural basis for this an of aeropin the with the (23Irving J.A. Steenbakkers P.J. Lesk A.M. Op den Camp H.J. Pike R.N. Whisstock J.C. Mol. Biol. Evol. 2002; 19: 1881-1890Crossref PubMed Scopus (105) Google Scholar). As the the presence of two that a in both its activity and stability (Fig. (23Irving J.A. Steenbakkers P.J. Lesk A.M. Op den Camp H.J. Pike R.N. Whisstock J.C. Mol. Biol. Evol. 2002; 19: 1881-1890Crossref PubMed Scopus (105) Google Scholar). antitrypsin these are and We that the form a between the and the while form a between the and the of the aeropin is by in the and presence of is a the presence of which the unfolding of aeropin (Fig. The presence of two first by the Arch. Biochem. Biophys. PubMed Scopus Google Scholar) in which aeropin with and the of the a no change in an of aeropin under a of aeropin molecule that are present in the native molecule. in which the with to the of the in aeropin stability and inhibitory the with at and has at and and has all the by serine (Fig. The of in the the the from the The inhibitory activity of the As shown in the inhibitory activity of both and significantly with in of an to the The of the the presence of and to have the in stability to with of °C and while a of which is to these data that of the at the RCL to the stability of the and with the at which results in a large of the of the of aeropin a (Fig. a of the at temperatures °C for both the and while the stable to temperatures °C (Fig. no observed which is with a of serpin between the stability of the in to have a remarkable resistance to thermal This the first biophysical characterization of a serpin from an to metastable and an efficient proteinase with the serpin mechanism of function. possesses a remarkable to both chemical and thermal and not polymerize in with or changes in pH as observed with other serpins (13James E.L. Bottomley S.P. Arch. Biochem. Biophys. 1998; 356: 296-300Crossref PubMed Scopus (74) Google Scholar, D.A. Carrell R.W. Nature. 1992; PubMed Scopus Google Scholar, S.T. A. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). unfolding of and other serpins has been shown to a first aggregation (13James E.L. Bottomley S.P. Arch. Biochem. Biophys. 1998; 356: 296-300Crossref PubMed Scopus (74) Google Scholar, E.L. Whisstock J.C. Bottomley S.P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, G.J. Bottomley S.P. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). All of the serpins to a which the of an intermediate ensemble J.A. Cabrita L.D. Rossjohn J. Pike R.N. Bottomley S.P. Whisstock J.C. Structure (Camb.). 2003; 11: 387-397Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, E.L. Whisstock J.C. Bottomley S.P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.H. J. 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Protein Sci. 2004; PubMed Scopus Google Scholar). mutations have also been demonstrated to the aggregation-prone intermediate in a A. B. C. J. J. Science. PubMed Scopus Google Scholar, B. R. A. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the stability of the aeropin native state an the biophysical of the presence of two these as unfolding and this to the for the covalent between and the strand However, of this a that of the between the and the RCL not significantly thermal stability. other with mesophilic serpins in that aeropin has a of and a of to the mesophilic This has been for other thermophilic proteins K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), and enhance stability the formation of S. R. Biochemistry. 2002; PubMed Scopus Google Scholar, R. J. Struct. Biol. 2007; PubMed Scopus Google Scholar). also a significantly number of and aeropin also significantly more residues the mesophilic serpins, of which are from the to predominantly the of these residues to form and the The metastability paradigm a balance between structural flexibility and inhibitory the of the is for effective inhibition. that is for a serpin to adopt a high native state stability and an As a result, conformational changes required for biological activity are while unfolding that misfolding are to the of native state stability with serpin inhibitory activity stability and in the of an in changes to the intermediate have also been This thus the that mesophilic serpins have shown to an increase in stability. as aeropin an unfolding that in not is with other serpins, has the of the intermediate ensemble and the between and activity.