EJ GOLDSMITH AND J MOlTONEN MINIREVIEW

Serpins: the uncut versionThe structure of active antithrombin, the first active serpin to be solved,

sheds new light on the conformational forms of this important class of inhibitor.

Structure 15 April 1994, 2:241-244

In Genesis, the serpent is described as "more subtilethan any beast of the field". The aptly named serpins(for seine protease inhibitors) seem capable of evermore 'subtile' and serpentine molecular meanderingswith each newly reported structure. These proteins (of-400 amino acids) have a seemingly flexible reactive-site loop able to adopt multiple conformations. Butupon protease binding, rather than undergoing facilecleavage, a tight-binding inhibitory complex is formed:apparently for the protease, "ye shall not eat of it, nei-ther shall ye touch it, lest ye die".

The first hint of this sinuous behavior came in 1984when the structure of the archetypal serpin, tal-pro-teinase inhibitor (l-PI), also called al-antitrypsin, wassolved in its cleaved and inactive form [ 1 ]: the two newtermini were separated by some 70 A, positioned at op-posite ends of the ellipsoidal molecule (Fig. la). Sub-sequently, the structures of two intact non-inhibitoryserpins were reported - a serpin homolog, ovalbumin[2,3] (Fig. lb), and a hyperstable 'latent' form, latentplasminogen activator inhibitor-1 (PAI-i) [4] (Fig. c).These three structures differ in the position and con-formation of the reactive-center loop. In the cleavedand latent forms, part of the reactive-center loop, be-tween residues P16 and P2 (counting backwards fromthe major recognition site at P1), form a central strand(4A) of the largest 13-sheet. In the substrate form, thereactive-center loop is formed into a helix at the top ofthe molecule, leaving sheet A with one less strand [51.

After this anticipation, the elusive serpin active confor-mation has now come into view. Two new reports, onefrom the Carrell laboratory in this issue [6] and onefrom the Hol laboratory [7], reveal the active confor-mation of antithrombin (Fig. d). The two studies arebased on similar crystals. Quite remarkably, both reportthe structure of an active molecule of antithrombin ina complex with an inactive molecule of antithrombin.The structures explain many biochemical properties ofactive serpins, while still leaving important questionsunanswered.

Although both structures were solved by molecular re-placement at relatively low resolution (3.0A [6] and3.2A [7]), thegreat similarity of the two determina-tions substantiates both studies. Most interestingly, thereactive-center loop is found to be partially insertedinto sheet A, making the conformation of sheet A very

unusual indeed. Residues P16 and P15 form 3-sheethydrogen-bonding interactions with strands 3A and 5A.The main chain leaves sheet A at P14 , but the sidechain Oy of serine P14 may form hydrogen bonds withthe main chain of neighboring strands (Fig. 2a). Theelectron density map reported by Schreuder et al. [7]shows clear electron density for the hairpin turn ob-served in cleaved serpins (from strand 5A to strand4A, residues P 18 -P 15). Carrell et al. [6] did not modelP1 6 -P 12 due to breaks in the density, but both they andSchreuder et al. observe most of the P1 l-P' 9 segmentof the reactive-center loop extending over the top ofsheet C. Extensive biochemical data have predicted thatstrand 4A is partially formed in the active molecule. Thesusceptibility to proteolysis by non-cognate proteasesof some but not all of the residues in the reactive-centerloop [8], and the non-inhibitory properties of serpinhomologs such as ovalbumin, mutant serpins [9], andserpins in complex with peptides corresponding to thefull length of strand 4A [10] suggest that strand 4A maybe partially inserted in an active serpin. However, thehydrogen-bonding pattern between strands 3A and 5Afor the remainder of the sheet was unexpected. The P-sheet is broken between strands 3A and 5A at positionscorresponding to P11 and P10 of an inserted strand 4A.

Both groups report the structure of an active copy ofantithrombin in complex with an inactive copy. Mostsurprisingly, this complex is formed by the insertionof the reactive-center loop of the active molecule (be-tween residues P7 and P2 ) as strand 1C of the inactivemolecule (Fig. 2c). Carrell et al. note that the observeddimer may be a model for the medically importantpolymerization behavior seen in the Z variant of Cl-PIand several other mutant serpins [ 11 ]. While Schreuderet al. report one intact and one cleaved antithrombinmonomer, Carrell et al. found no evidence for cleavage(on the basis of amino-terminal sequence analysis) andsuggest that both molecules are intact, one possess-ing activity and the other adopting an inactive confor-mation similar to latent PAI-1. Their conclusion seemslikely to be right. Although electron density is lackingfor the reactive-center loop of the latent molecule be-tween P' 3 and P' 10 in both determinations, the place-ment of residues P2 to P' 2 in both structures suggestsa latent rather than a cleaved conformation. Electrondensity may be lacking because a stabilizing salt bridge,present in latent PAI-I, is missing in antithrombin. The

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EJ GOLDSMITH AND MOTTONEN MINIREVIEW

242 Structure 1994, Vol 2 No 4

Fig. 1. Ribbon diagrams of observedserpin conformations. (a) Cleaved ct-proteinase inhibitor [17]. (b) Ovalbumin121. (c) Latent PAI-1 [4]. (d) Antithrom-bin [6,71. Corresponding residues of thereactive-center loop between P16 andP'9 are colored in orange. In (a), sheetsA and C are in purple. (Figures weredrawn using Molscript [18].)

slow formation of a latent molecule of antithrombinprovides a plausible explanation for the observed slowrate of crystal growth [7]. Crystals from both laborato-ries, when dissolved, possessed some inhibitory activityagainst protease. This is presumably a property of themolecule with the exposed reactive-center loop.

The interaction of the reactive-center loop with sheet Cis reminiscent of the binding mode of small protein in-hibitors. For example, in the case of bovine pancreatictrypsin inhibitor, residues P'4-P 2 form hydrogen-bond-ing interactions with the inhibited protease [12]. Thissimilarity in the binding mode was anticipated by muta-genesis studies on PAI-1-inhibited tissue plasminogenactivator [13]. An exception is the P1 arginine which

is found not pointing away, but arched back towardsthe core of the protein, nearly forming a salt bridgewith Glu255 of the inhibitor. Carrell et al speculatethat the observed salt bridge may be present in thefree inhibitor, and may play a role in the activation ofantithrombin by heparin.

How relevant is this structure to that of a serpin in anactive state, bound to and forming nearly irreversiblecomplexes with protease? Schreuder et al. [7] rea-sonably believe that the reactive-center loop is un-likely to adopt the well-defined extended conforma-tion observed when free in solution, but that it rep-resents a working model for an active serpin boundin a complex. They used this conformation to model

(I

al-PI

(c) (d)

Latent PAI-1

Serpins Goldsmith and Mottonen 243

Fig. 2. The antithrombin complex.(a) The complex: active molecule, bodyblue and reactive-center loop orange;the latent molecule, body green, react-ive-center loop red. (b) Close up of thepartially inserted strand 4A in the ac-tive molecule. Coordinates were pro-vided by Carrell et al. 16]; residues be-tweeen P16 and P12 were modeled ac-cording to the description of Schreuderet al. [71 and displayed as alanines, al-though the actual seqeunce is EGSEA.(c) Close up of the interaction of thereactive-center loop inserted as strand1C in the latent molecule.

antithrombin's interaction with thrombin. Carrell et al[6] suggest that the conformation of strand 4A thatthey observe may be peculiar to this complex. Theypropose that an unbound inhibitor might have reducedor no formation of strand 4A, like ovalbumin, whilea tight complex with a protease might involve furtherinsertion of the reactive-center loop and conversion tostrand 4k As evidence for the latter proposal, they notethat the A-sheet is open to Plo. A more fully formedstrand 4A might explain the immunological evidencethat antithrombin-thrombin complexes have an epi-tope that is present in cleaved antithrombin but ab-sent from active uncomplexed antithrombin [14]. Themolecular details underlying this conformational trans-ition have yet to be clearly delineated.

Serpins are the major protease inhibitors found inplasma and appear to have evolved multiple confor-mational states to allow for a diversity of functions.Serpin-protease complexes are unusually stable, re-quiring strong denaturants to be dissociated. Further-more, the inhibitors are single-use molecules: on dis-sociation from the protease they are cleaved and in-active as inhibitors. The folding pathway for serpinsmust be unusual, since the active conformation is un-stable relative to the latent form [15]. Thus, the ac-tive form is reminiscent of a late stage folding in-termediate that possesses unstructured loop regions

L16J. This kinetically-controlled activity may allow theenergy of folding to be used to achieve irreversiblecomplex formation. Without clear structural data for aserpin-protease complex, the folding pathway is oneof several problems that remain to tempt those seekingknowledge about the serpins.

References1. Loebermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R.

(1984). Human cl-proteinase inhibitor. J. Mol Biol 177,531-556.

2. Stein, P.E., Leslie, AG.W., Finch, J.T., Tumell, W.G., McLaugh-lin, PJ. & Carrell, R.W. (1990). Crystal structure of ovalbuminas a model for the reactive centre of serpins. Nature 347,99-102

3. Wright, H.T., Qian, H.X. & Huber, R (1990). Crystal structureof plakalbumin, a proteolytically nicked form of ovalbumin.J. Mol Biol. 213, 513-528.

4. Mottonen, J., et al, & Goldsmith, EJ. (1992). Structural basisof latency in plasminogen activator inhibitor-1. Nature 355,270-273.

5. Stein, P.E. and Chothia, C. (1991). Serpin tertiary structuretransformation. j MoL Biol. 221, 615-621.

6. Carrell, R.W., Stein, P.E., Fermi, G. & Wardell, M. (1994).Biological implications of a 3A structure of dimeric anti-thrombin. Structure 2, 257-270.

7. Schreuder, HA, et al, & Hol, W.GJ. (1994). The intact and

244 Structure 1994, Vol 2 No 4

cleaved human antithrombin III complex as a model for

serpin-proteinase interactions. Nature Struct. Biol. 1, 48-54.8. Mast, AE., Enghild, JJ. & Salvesen, G. (1992). Conformation

of the reactive site loop of a -proteinase inhibitor by limitedproteolysis. Biochemistry 31, 2720-2728.

9. Skriver, K., et al, & Bock, S.C. (1991). Substrate properties ofC1 inhibitor Ma (alanine 434 glutamic acid). J Biol. Chem266, 9216-9221.

10. Schulze, A.J., Frohnert, P.W., Engh, R.A. & Huber, R. (1992).Evidence for the extent of insertion of the active site loop

of intact al-proteinase inhibitor in 3P-sheet A Biochemistry31, 7560-7565.

11. Lomas, D.A., Evans, D.L.I., Finch, J.T. & Carrell, R.W. (1992).The mechanism of Z al-antitrypsin accumulation in the liver.

Nature 357, 605-607.12. Papamokos, E., et al., & Laskowski, MJr. (1982). Crystallo-

graphic refinement of Japanese quail ovomucoid, a Kazal-type

inhibitor, and model building studies of complexes with ser-

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13. Madison, E.L, Goldsmith, EJ., Gerard, R., Gething, MJ. &Sambrook, J.F. (1989). Serpin-resistant mutants of humantissue-type plasminogen activator. Nature 339, 721-723.

14. Bjork. ., Nordling, K. & Olson, S.T. (1993). Immunologic evi-dence for insertion of the reactive-bond loop of antithrombininto the A 3-sheet of the inhibitor during the trapping oftarget proteinases. Biochemistry 32. 6501-6505.

15. Carrell, R.W.. Evans, D.L.I. & Stein, P.E. (1991). Mobile reactive centre of serpins and the control of thrombosis. Nature

353, 576-578.16. Redfield, C.. Smith, R.A.G. & Dobson, C.M. (1994). Structural

characterization of a highly-ordered 'molten globule' at lowpH. Nature Struct. Biol 1, 23-29.

17. Huber. R. & Bode, W. (1978). Structural basis of the activa-tion and action of trvpsin. Accounts Chem. Res. 11, 114-122.

18. Kraulis, PJ. (1991). MOLSCRIPT: a program to produce bothdetailed and schematic plots of protein structures. J ApplCrystallogr. 24, 946-950.

EJ Goldsmith and J Mottonen, Department of Biochem-istry, The University of Texas Southwestern Medical Cen-ter at Dallas, 5323 Harry Hines Blvd., Dallas, Texas 75235-9038, USA.