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nature structural biology • volume 7 number 4 • april 2000 267

When you are wounded, thrombin and ahost of other blood clotting proteins rushto the rescue, stemming the flow of blood.Once repaired, thrombin rapidly retreatsto ensure that the blood vessels stay clear.This delicate balance between coagulationand anticoagulation is key to hemostasisand to designing drugs that act specificallywithin a cascade of proteases. The struc-tural basis of this balance is the focus oftwo independent papers appearing inNature and Nature Structural Biology. Theregulation of thrombin by thrombomod-ulin involves an induced fit in an epi-dermal growth factor (EGF) domainaccording to Komives and colleagues1. Acrystal structure by Bode and coworkers2

depicts thrombin sitting on a saddle ofEGF domains. Because the structure ofthrombin is basically the same whether ornot it is bound to the EGF domains, theidea that thrombomodulin allostericallyalters thrombin specificity2 has been putto rest. Instead, thrombomodulin’s spe-cialized EGF modules block clot-promot-ing substrates from accessing thrombin’sactive site while serving as an adapter thatrecruits other, anticoagulant substrates.

Thrombin is a serine protease whosespecificity determines the fate of theblood coagulation cascade. The last in achain of membrane-bound factors andcofactors (Fig. 1), thrombin cleaves plas-ma fibrinogen into self-polymerizing fib-rin monomers3. Thrombin also activatesfactor XIII to convert fibrin into an insol-uble network that is plugged by circulat-ing platelets. However, once the vasculardamage is contained, thrombin cleavesand activates protein C instead4. ProteinC shuts down the coagulation cascade bydegrading two upstream factors requiredto activate thrombin. Residual thrombinis rapidly inactivated, and fibrin clot for-mation halts. Thrombin also inhibitsfibrinolysis by activating thrombin acti-vatable fibrinolysis inhibitor (TAFI)5. Thetide turns, blood coagulation is supplant-ed by anticoagulation, and the woundheals.

Thrombin’s ability to activate protein Cand TAFI depends on the presence ofthrombomodulin. This glycoprotein wasidentified as a modulator of thrombinspecificity6, and its expression on the sur-face of endothelial cells is tightly regulatedby inflammatory cytokines (reviewed inref. 7). Thrombomodulin features a stringof six EGF repeats between an N-terminallectin-like region and a C-terminal trans-membrane region. The fifth EGF repeat(EGF5) is essential for thrombin bindingand possesses an unusual disulfide bond-ing pattern8. In the classical EGF fold cys-teines 1–3, 2–4, and 5–6 are linked, but inEGF5 cysteines 1–2, 3–4, and 5–6 arelinked in order. The sixth EGF repeat(EGF6) provides a 10-fold increase inthrombin affinity9, while the fourth repeat(EGF4) is needed to enhance protein Cactivation ∼ 1,000-fold7. However themechanism by which these small, flexible

domains conspire to switch thrombin’sspecificity has remained a mystery.

The new structures1,2 reveal the unex-pected nature of thrombomodulin’s cun-ning switch. Thrombomodulin does notretool thrombin’s active site, contrary to theallosteric mechanism suggested by muta-genic, fluorescence, and spin labeling stud-ies. Only minor differences are found inthrombin’s structure in and out of theEGF456 saddle2. The pair of six-stranded β-barrels that constitute thrombin’s fold sitsundisturbed. Small changes are seen in sev-eral exposed loops including the 60-loop, a point of insertion that bordersthe active site region. Surprisingly, catalyticresidues are essentially unmoved and mani-fest similar interactions with boundinhibitors. The allosteric theory has died asudden death.

Thrombomodulin alters thrombin’sspecificity in two ways. First, thrombomod-

The plot thickens: How thrombinmodulates blood clottingMichael Overduin and Tonny de Beer

The tide of the blood coagulation cascade turns by a switch in thrombin specificity. Two new structures revealhow thrombomodulin’s EGF domains flip the switch by blocking old substrates and docking new ones.

Fig. 1 The blood coagulation cascade involves proteolytic activation of a series of membranebound proteases. Activated thrombin cleaves fibrinogen into fibrin, which is crosslinked into clotsby the transglutaminase activated factor XIII. Anticoagulation occurs when thrombomodulin’s EGFdomains bind thrombin, initiating feedback regulation of factors V and VIII by activated protein C(aPC) and clot stabilization by thrombin activatable fibrinolysis inhibitor (TAFI). Inactive and activeforms of enzymes are depicted as full and partial circles, respectively, and are designated by romannumerals without and with an ‘a’, respectively. Membrane association domains are shown as tails.

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ulin’s EGF56 tandem occludes exosite I (Fig.2a), a basic patch on thrombin’s surface thathelps to bind clotting factors V and VIIIand fibrinogen2. Second, thrombomod-ulin’s EGF4 domain serves as a prostheticarm that grabs anticoagulant substrates likeprotein C, presenting them for cleavagedespite their non-optimal sequences.Rather than changing the way the enzymeacts, thrombomodulin blocks access by theusual substrates and presents new sub-strates in a way that facilitates cleavage.

Thrombomodulin’s ability to presentthrombin with novel substrates relies oninteractions between its EGF domains.The linker between EGF4 and EGF5 is func-tionally critical. The former domain posi-tions protein C, and the latter latches ontothrombin. An unanticipated perpendicu-lar angle is found between the two EGFdomains in the thrombin–thrombomod-ulin complex (Fig. 2a). EGF4 angles awayfrom rest of the complex and is tethered tothe linker by several hydrogen bonds2. Inthe uncomplexed state EGF4 exhibitssomewhat different contacts with EGF5,whose solution structure is relatively dis-ordered (Fig. 2b,c)1. For example, contactsbetween Ala 377 of EGF4 and the N-acetylglucosamine moiety linked to Asn 391 ofEGF5 are found only in the NMR struc-ture, and the two domains appear to be incloser proximity in solution.

More surprises lurk in the hydrophobiccore. In the thrombin–EGF456 complex2 thecore of the EGF5 domain anchors linkerresidue Met 388. A similar interaction isseen for this functionally essential residuein the uncomplexed tandem EGF45

(ref. 1). This contrasts with the disorderedand exposed position of Met 388 in anEGF5 fragment in which hydrophobicresidues that play roles in thrombin bind-ing are buried10. Interestingly, the confor-mation of EGF5 is more dynamic whenattached to EGF4 than when not attached toEGF4. Specifically, the EGF5 domainexhibits lower heteronuclear 1H-15N NOEvalues, more sparse NOE density, andlower structural convergence in the contextof the EGF45 tandem1. Thus, in the contextof EGF45, Met 388 inserts into EGF5’s coreand triggers a flexible state that more close-ly resembles the thrombin-bound state interms of core packing1. This rearrangementaccounts for the considerable affinity (KD =120 nM) of EGF45 for thrombin9.

Could the dynamic character of EGF5

allow it to change more readily into ashape complementary to thrombin’s bind-ing site? When thrombin binds EGF45,chemical shifts are perturbed throughoutEGF5, indicating major conformationalchanges, and hence, induced fit1. Indeed,in the crystallized complex, the conforma-tion of EGF5 is more extended and is well

268 nature structural biology • volume 7 number 4 • april 2000

defined in terms of electron density2. Inparticular, the β2-β3 hairpin of EGF5

extends to contact thrombin’s active siteregion, in contrast to its more compactstructure in the free state (Fig. 2).

The role of the EGF6 domain also needsto be considered. The apparently fragilestructure of EGF5 is fortified by the pres-ence of its neighbor, EGF6, whose classicalEGF fold is augmented by an extra C-ter-minal strand2. EGF6 stabilizes EGF5

through hydrophobic and calcium-mediat-ed interactions. For example, the β2-β3loop of EGF6 packs against Tyr 413, anEGF5 residue required for cofactor activity.In addition, a calcium ion critical for cofac-tor activity is bound between EGF5 andEGF6 with an affinity of 2 µM11. Residuesfrom the two EGF domains coordinate cal-cium2, and calcium also stabilizes the β-sheet structure of these EGF domains inthe thrombin-free state11. Thus, structuralconnections between thrombomodulin’sEGF domains are maintained in both thethrombin-bound and free states.

An unforeseen interface is presentbetween thrombin and thrombomodulin’sY-shaped saddle. A largely electrostaticinteraction was anticipated betweenthrombin and EGF56 based on a crystalstructure of thrombin bound to an acidicEGF5 peptide via its basic exosite I12.However hydrophobic interactions domi-nate the 900 Å2 interface2. Thrombin’sbasic side chains interact with solvent, andsome of thrombomodulin’s acidic sidechains coordinate calcium. Nonetheless,the interacting thrombin and EGF56 sur-faces complement each other in terms ofelectrostatic potentials. A second basicpatch on thrombin’s surface interacts witha negatively charged chondroitin sulfatemoiety located between EGF6 and throm-bomodulin’s transmembrane region.Consequently, docking of the two proteinsdoes rely on electrostatic attractions.However, the complex is held together byhydrophobic interdigitations that involve alocal induced fit in thrombin’s exosite I andEGF56, consistent with the induced fit ofEGF5 inferred by Komives and colleagues1.

A tantalizing speculation of how throm-bomodulin stuffs protein C into thrombin’sactive site is offered by Bode and cowork-ers2. This model represents the entire pro-tein machinery responsible for initiatinganticoagulation, and explains how proteinC is cleaved despite the fact that it is nor-mally a poor substrate. A crystal structureof activated protein C13 was docked ontothe thrombin–EGF456 complex, and proteinC’s cleavage site was modeled into the activesite in a substrate-like manner. Protein C

Fig. 2 Structure of thrombomodulin’s EGF domains in the thrombin-bound and free states. a, A rib-bon diagram of thrombin in complex with thrombomodulin’s EGF456 domains1. The heavy and lightchains of α-thrombin are shown in green, and exosite I is shown in red. An irreversible inhibitor, L-Glu-Gly-Arg chloromethyl ketone, is bound to the active site and is shown as a space filling model.The fourth, fifth, and sixth EGF domains of the thrombomodulin fragments are labeled and shownin purple, dark and light blue, respectively. Calcium and sodium ions are depicted as orange and pur-ple spheres, respectively. Disulfide linkages in the EGF modules are drawn as yellow sticks. ‘N’ and ‘C’designate N- and C-termini of the thrombomodulin fragments. b, The ensemble of ten solutionstructures of the thrombin-free EGF4 and EGF5 tandem (EGF45)2 are oriented and colored as in (a). Thebackbone traces of EGF4 residues are superimposed. c, A ribbon model of the EGF45 tandem with abackbone structure most similar to the thrombin-bound state is oriented and colored as in (a).

a b

c

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history

abuts an EGF4 surface rich in functionallycritical residues. An electrostatic interactionis predicted here between the acidic face ofEGF4 and basic residues within the proxi-mal “37” and “70” loops of protein C’sactive and calcium binding sites, respective-ly. This interface can accommodate a bridg-ing calcium ion at the position of a sodiumion bound to EGF4 (Fig. 2a), in agreementwith an earlier suggestion14.

The model of the ternary complex ofthrombin, EGF456, and protein C predictsadditional interactions. For example,thrombomodulin’s acidic EGF3 domaincan be modeled in near Lys 62 and Lys 63 ofprotein C. These two exposed residues con-tribute significantly to the activation ofprotein C by thrombomodulin-boundthrombin15. A similar interaction may beparticularly relevant for the recruitment ofTAFI, activation of which requires EGFrepeats lying N-terminal to thrombomod-ulin’s EGF4 domain16. Interactions on thesurface of endothelial cells can also beenvisaged. A distance of 50 Å between thecatalytic site and membrane is predicted bythe model, and compares well with experi-mental measures7. The ternary complex isanchored to the membrane by protein C’sN-terminus and thrombomodulin’s trans-membrane region. By independentlyanchoring protein C and thrombin inendothelial membranes, substrate and

Art Horwich worked closely with Paul Siglerat Yale University on the project to determinethe structure of the GroEL chaperonin1. Here,he recounts memories of how the collabora-tion began and evolved.

“I first met Paul in 1989. My group hadjust published work in collaboration withUlrich Hartl and Walter Neupert showingthat Hsp60 in mitochondria was requiredfor folding/assembly of newly-importedmitochondrial proteins2,3. In the sameyear, George Lorimer’s group reconstitut-ed a chaperonin-mediated folding reac-tion in vitro with purified bacterial GroEL,GroES, and ATP4. Chaperonins were look-ing more and more interesting, but werealso pretty mysterious. It seemed that a

major way forward would be to obtainstructural information.

“I went across campus to visit Paul on acompletely different matter, but our con-versation very rapidly turned to chaper-onins. He was immediately fascinated bytheir activity and by what the structure ofsuch a machine might be. He corralledZbyszek Otwinowski and articulated tohim the amazing symmetries that must bepresent in the GroEL molecule. Zbyszekhimself became rapt but was worried thatthe available computing power might notbe able to handle something the size ofGroEL. But he said that he was ready tothink further, if and when we could devel-op decently diffracting crystals. As it turned

nature structural biology • volume 7 number 4 • april 2000 269

enzyme are concentrated in a two dimen-sional plane. Furthermore, they are posi-tioned at a comparable distance from themembrane surface in order to interact pro-ductively.

The take home message of these newstudies is the most straightforward —position can override specificity. Ratherthan tinker with the specificity determi-nants in thrombin’s active site, thrombo-modulin positions protein C for cleavageand sterically blocks access by other sub-strates. Protein C’s activation site remainsa poor substrate, and further structuresare needed to resolve how it is accommo-dated by thrombin’s active site.Nonetheless, thrombomodulin overpow-ers these local incompatibilities by pre-senting protein C in such a fashion thatthrombin cannot resist cleaving it.

Finally, the new structures offer newopportunities for drug design. The devel-opment of several generations of thrombininhibitors has been spurred on by previousstructural studies17. Major clinical indica-tions of such anticoagulants include car-diovascular disorders and thrombosis.However, the therapeutic utility of theseagents has been hampered by the lack ofsufficient specificity and correspondinglycomplex pharmacologic profiles. Themechanism by which thrombin achievesspecificity has now been established, and

new interfaces are available for the designof improved therapeutic agents.

AcknowledgmentsWe thank P. Fuentes-Prior and M.J. Wood forproviding atomic coordinates, and J. Mamay forhelping with figure preparation.

Michael Overduin and Tonny de Beer are inthe Department of Pharmacology andBiomolecular Structure Program, Universityof Colorado Health Sciences Center, Denver,Colorado 80220, USA. Correspondenceshould be addressed to M.O. email:Michael.Overduin@UCHSC.edu

1. Wood, M.J., Sampoli Benitez, B. A. & Komives, E. A.Nature Struct. Biol. 7, 200–204 (2000).

2. Fuentes-Prior, P. et al. Nature in the press (2000).3. Ferry, J.D. & Morrison, P.R. J. Am. Chem. Soc. 69,

388–399 (1947).4. Kisiel, W., Ericsson, L.H. & Davie, E.W. Biochemistry

15, 4893–4900 (1976).5. Bajzar, L., Manuel, R. & Nesheim, M. E. J. Biol.

Chem. 270, 14477–14484 (1995).6. Esmon, C.T. & Owen, W.G. Proc. Natl. Acad. Sci. USA

78, 2249–2252 (1981).7. Esmon, C.T. FASEB J. 9, 946–955 (1995).8. White C.E. et al. Proc. Natl. Acad. Sci. USA 93,

10177–10182 (1996).9. White, C.E. et al. Protein Eng. 8, 1177–1187 (1995).

10. Sampoli Benitez, B.A., Hunter, M. J., Meininger, D.P. & Komives, E.A. J. Mol. Biol. 273, 913–926 (1997).

11. Light, D.R. et al. Eur J. Biochem. 262, 522–533 (1999).12. Mathews I.I., Padmanabhan, K.P., Tulinsky, A. &

Sadler, J.E. Biochemistry 33, 13547–13552 (1994)13. Mather, T. et al. EMBO J. 15, 6822–6831 (1996).14. Zushi, M. J. Biol. Chem. 266, 19886–19889 (1991).15. Knobe, K.E. et al. Proteins 35, 218–234 (1999).16. Nesheim, M. et al. Thromb. Haemost. 78, 386–391

(1997).17. Fareed, J. et al. Semin. Hematol. 36, 42–56 (1999).

Working with Paul Sigler Art Horwich

out, time was on his side, because it took usfour years, with our two groups workingsteadily together, to generate such crystals.

“The early workhorses were AndrzejJoachimiak, a senior scientist in Paul’sgroup, and Kerstin Braig, a student in mygroup. They first tried working with ther-mophilic bacterial versions of GroEL,thinking these might be better behavedthan the E. coli molecule, about which wehad heard only bad news. We thought thatmaybe the thermophilic versions would bebetter ordered — but no luck. Next we triedseveral truncated molecules engineered inmy lab that retained ATPase activity — alsonot successful. But by this time, it was get-ting easier to produce gram amounts ofclean protein that allowed us to set updozens of trays at once. I remember Andrzejmarveling at an expression construct thatproduced GroEL at >95% soluble bacterialprotein, as the only band on a gel!

“Meanwhile, Paul’s group had movedacross campus to be our neighbors in the

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