Structural Basis of the Adaptive Molecular Recognition by MMP9

Structural Basis of the Adaptive MolecularRecognition by MMP9

Hyunju Cha1, Erhard Kopetzki2, Robert Huber1, Martin Lanzendorfer2

and Hans Brandstetter1*

1Max-Planck-Institutfur Biochemie, AbteilungStrukturforschung, D-82152Martinsried, Germany

2Roche Diagnostics GmbHPharma Research, D-82372Penzberg, Germany

Matrix metalloproteinase (MMPs) are critical for the degradation of extra-cellular matrix components and, therefore, need to be regulated tightly.Almost all MMPs share a homologous C-terminal haemopexin-likedomain (PEX). Besides its role in macromolecular substrate processing,the PEX domains appear to play a major role in regulating MMP acti-vation, localisation and inhibition. One intriguing property of MMP9 isits competence to bind different proteins, involved in these regulatoryprocesses, with high affinity at an overlapping recognition site on its PEXdomain. With the crystal structure of the PEX9 dimer, we present thefirst example of how PEX domains accomplish these diverse roles. BladeIV of PEX9 mediates the non-covalent and predominantly hydrophobicdimerisation contact. Large shifts of blade III and, in particular, blade IV,accompany the dimerisation, resulting in a remarkably asymmetrichomodimeric structure. The asymmetry provides a novel mechanism ofadaptive protein recognition, where different proteins (PEX9, PEX1, andTIMP1) can bind with high affinity to PEX9 at an overlapping site. Finally,the structure illustrates how the dimerisation generates new properties onboth a physico-chemical and functional level.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: activity regulation; b-propeller; hemopexin; induced fit; MMP9*Corresponding author

Introduction

Matrix metalloproteinase (MMPs) are criticalfor the degradation of extracellular matrix com-ponents. They are involved in a multitude ofphysiological processes, like tissue remodelling orwound healing, and pathological processes, suchas arthritis, inflammation and tumour cellmetastasis.1 – 3 Consequently, their activity is regu-lated on several levels, including gene expression,zymogen–enzyme activation, and natural inhi-bition by endogenous inhibitors of MMPs(TIMPs). MMPs are mosaic proteins containing anN-terminal pro-domain, the catalytic Zn2þ proteasedomain and the C-terminal haemopexin-likedomain (PEX). Gelatinases A and B (MMP2 andMMP9) form a subgroup within the MMP familywith preferential substrate specificity towards typeIV collagen.4 Both enzymes have three FN-II type

domains interspersed within their catalyticdomain. Moreover, their PEX domains (PEX2 andPEX9) are able to form high-affinity complexeswith the C-terminal domains of TIMP2 andTIMP1, respectively. MMP9 is found in largeamounts in the granules of neutrophils, which pro-vide a first line of defence to the immune system.5

The importance of MMP9 for inflammatorydiseases relates to their significance for cellmigration.6 In addition, MMP9 is able to degradethe myelin basic protein, thereby releasingencephalitogens, which are thought to contributeto the pathogenesis of multiple sclerosis.7,8 Finally,MMP9 was shown to coordinate and effect mul-tiple events involved in the process of epithelialregeneration.6

Structures of PEX domains determined so farinclude the full-length structures of MMP19 andpro-MMP2;10 the isolated PEX domain of humanMMP2 (denoted PEX2) in two different crystalforms;11,12 and human PEX13.13 In addition,structures of the C-terminal domain of rabbitserum haemopexin and of the haem–haemo-pexin complex from Oryctolagus cuniculus weredetermined.14,15

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

E-mail address of the corresponding author:[email protected]

Abbreviations used: MMP, matrix metalloproteinase;PEX, haemopexin-like domain; TIMP, endogenousinhibitor of MMP.

doi:10.1016/S0022-2836(02)00558-2 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 320, 1065–1079

Dimerisation appears to provide a major mecha-nism to regulate MMP activity. The dimerisationof PEX14, the PEX domain of MMP14(MT1-MMP), is a prerequisite for the efficientactivation of proMMP-2.16 There are conflictingdata on the formation of the PEX142 homodimer,however.17 Moreover, (pro)MMP9 is known toexist in both a monomeric and a dimeric state,which is reduction-sensitive and, therefore,believed to be disulphide-linked.18 In particular,the dimerisation decreases the rate of activation ofproMMP9 by stromelysin (MMP3) significantly.18

Furthermore, MMP9 is able to bind MMP1 to forma cooperative complex with extended substratespecificity against both gelatin and fibrillar type Icollagen.19 There are conflicting data concerningwhether dimeric MMP92 is able to form complexeswith TIMP1 to form a tetrameric MMP92 –TIMP12

complex18 or whether the MMP9–TIMP1 complexis incompatible with PEX9 homodimerisation.19

Here, we show that the dimerisation of MMP9is mediated by PEX9, which forms SDS-stablehomodimers. We present the crystal structureof the PEX92 dimer and show that this dimeris non-covalent, albeit reduction-sensitive. Thecrystal structure of PEX9 further reveals novelmechanisms that explain its adaptive proteinrecognition.

Results

In vitro folded PEX9 forms a non-covalent,SDS-stable homo-dimer

ProMMP9 is known to form a reduction-sensi-tive homodimer. Therefore, it was suggested thatthe dimerisation is mediated by a disulphide bondinvolving Cys468 and/or Cys674, both of whichare unique to MMP9.18,19 In an attempt to investi-gate the role of the PEX domain of MMP9 (PEX9),we have expressed PEX9 in Escherichia coli asinclusion bodies. We were able to oxidatively foldand purify PEX9 to apparent homogeneity at ayield of approximately 50 mg per litre of cellculture. Gel-filtration chromatography results in abroad peak corresponding to a size of approxi-mately 40 kDa, suggesting that the protein formspredominantly dimers. When run on a non-reducing SDS-12% (w/v) polyacrylamide gel, theprotein migrates as a monomer and a dimer(Figure 1, lane a). The 40 kDa band is not presenton the gel when run under reducing conditions(Figure 1, lane b). The protein is soluble at concen-trations .1 mg/ml only in the presence of 10 mMarginine. In the absence of arginine, the dimericform of the protein is completely SDS-stable (datanot shown). Taken together, our data indicate thatPEX9 mediates the dimerisation of MMP9. Whilewe confirm its critical dependence on reducingagents, the dimer formation is reversible and isnot caused by intermolecular disulphide linkages.

PEX9 adopts a four-bladed b-propeller structure

The overall structure of PEX9 is displayed inFigure 2, and adopts the four-bladed b-propellerfold seen in all previously determined structuresof the C-terminal MMP domains.9,11 – 13 Thedisulphide bond between the conserved Cys516and Cys704 connects blade I with blade IV, and isobserved in the corresponding position in allhomologous structures reported so far. This intra-molecular covalent linkage is critical for the struc-tural integrity of all four-bladed b-propellers. Thepresence of reducing agents will cleave thedisulphide bond and presumably disruptthe b-propeller structure, thus explaining thereduction sensitivity of the proMMP9 dimer.

C-terminal blade mediates the mainlyhydrophobic dimer contact

The dimerisation of MMP-9 was proposed to bedue to intermolecular disulphide bonding.18,19

However, the remaining free cysteine residuewithin the PEX9 domain, Cys674, is buried and,consequently, not involved in the dimerisation ofPEX9. Instead, the crystal structure shows that thedimerisation is due to non-covalent interactions ofthe two PEX9 domains, referred to as molecules Aand B. The two propeller axes are oriented roughly

Figure 1. Non-reducing and reducing SDS/12% (w/v)polyacrylamide gel indicating the soluble fraction, andthe dependence of the dimerisation on the intramolecu-lar disulphide bond. Lane a shows that much of PEX9 isin the monomeric state, due to the presence of 10 mMArg, while it is completely dimeric in its absence (datanot shown). With the addition of DTT (lane b), nodimeric PEX9 can be seen by SDS-PAGE. Moreover, weassume that the two bands at around 21 kDa, withidentical N-terminal sequence, represent two differentfolding conformations of monomeric PEX9. The faster-migrating band should be a more compactly foldedspecies, explaining its absence under reducing con-ditions (lane B).

1066 Mechanism of MMP9 Dimerization

antiparallel to each other. The C-terminal regionsof the propeller, in particular blade IV, form mostof the dimer contacts (Figure 2(a) and (b)). Theresidues contributing to the dimer interface areconserved through different MMP9 sequences (seeFigure 4), thus confirming the correct assignmentof the physiological dimer from the crystal lattice.Moreover, the Ta6Br12 cluster used in MAD phasingbinds exactly onto the dimer interface (Figure 2),and the dimer builds the crystallographic asym-metric unit of the crystal.

Molecules A and B are related to each other by a1728 rotation along an axis running approximately

perpendicular to the propeller axes. This axis runsin the vertical direction in Figure 2(a), and out ofthe plane in Figure 2(b). While the b sheets of theblades IV of molecules A and B are approximatelyparallel with each other, these strands do notextend the characteristic hydrogen bonding patternto form an eight-stranded sheet. The detailed inter-action is shown in Figure 3(a) and listed as a matrixin Table 1. This analysis reveals a predominantlyhydrophobic interaction.

The dimer is asymmetric

Molecules A and B deviate from each other withan overall root-mean-square (r.m.s.) deviation of1.3 A (0.70 A for back-bone atoms). This deviationcan be further restricted to blade IV of the molecule(Asp643-Asp707), which deviates by 1.53 A (1.02 Afor backbone atoms), whereas the r.m.s. differenceof the residual N-terminal part (Asp513-Val642) isonly 1.17 A (0.41 A). This deviation from the strict2-fold symmetry is caused by the contacts involvedin dimer formation. The interaction matrix alsoreflects the deviation (Table 1). It reveals a non-symmetric interaction pattern and involves mostlyhydrophobic residues, shown in Figure 3(a).Notably, there is only one salt-bridge contributingto the PEX9 dimer contact; namely, the C terminusof PEX9(A) with the side-chain of R677 ofPEX9(B). The contacting residues with at least fourinterdomain contacts are highlighted in Figure 4.They are generally conserved within MMP9through different organisms. Exceptions from thesequence conservation are Pro655 (Ser or Asn) andArg685 (Gln or His), with the deviating sequencegiven in parentheses. Both interaction sites arespecial in contributing only to molecule B, but notto the interface of molecule A. Moreover, Arg685forms an intra-domain hydrogen bond withGln690 and interacts with Leu659 of the dimer-related molecule A only via the aliphatic part of itsside-chain. The side-chains of glutamine or histi-dine may exhibit this hydrophobic interaction.Similarly, the contact of Pro655 with Gly656 ofmolecule A may be substituted by other aminoacids.

Repositioning of blades III and IV

The comparison with PEX2 and structurallyrelated PEX domains reveals a significant shift ofblade III and IV of PEX9 and is shown in Figure 5.This blade repositioning is observed in bothmolecule A and B. The relative vectorial shifts aresummarised in Table 2. These data show that therelative shift of blades III and IV is predominantlyalong the propeller (z) axis and amounts to morethan 1 A, when compared with PEX2. Since theshift direction of blades III and IV is opposite toeach other, their relative displacement along thepropeller axis is more than 2 A. In addition,we observe a movement within the plane of thepropeller (DX, DY), which leads to a widening of

Figure 2. (a) Overall view of the homodimeric PEX9structure in ribbon representation. The rainbow colour-coding indicates the sequential architecture, with blade Iin blue and blade IV in red. The view is perpendicularto the local 2-fold axis, which runs in the vertical direc-tion. The binding site of Ta6Br12 is formed by the Ctermini at the interface of both domains. Note, that Naþ

is bound to PEX9(A) only. The disulphide bonds (yellow)connecting blade I and blade IV are shown in a ball-and-stick representation. The ribbon representations wereprepared by using the programs MOLSCRIPT43 andRaster3D.44 (b) Overall view with identical colour-codingas in (a), but rotated 908. The view is now along themolecular 2-fold axis.

Mechanism of MMP9 Dimerization 1067

the propeller channel. The opposing blades I andIII contribute to this movement significantly, asevidenced by their opposite shifts DY (Table 2).

No calcium is bound to PEX9

There are conflicting data concerning the exactnature of the conserved metal ions bound within

the propeller channel of the previous structures.There is disagreement within PEX2, where theinternal metal ions were interpreted from thebottom of the channel to its top as: Ca2þ, Cl2,Naþ;11 and Ca2þ, Cl2, Ca2þ.12 Additionally, there isdisagreement on the metal ion located at the bot-tom entrance of the b-propeller, which was inter-preted as calcium in the X-ray structure of PEX1,9

and in both independently determined structuresof PEX2,11,12 and PEX13.13 By contrast, the metalion at this position was interpreted as Naþ inboth haemopexin structures.14,15 In the structure ofPEX13, a fourth metal ion is bound in the channelof the propeller,13 whereas an additional phosphategroup binds into the channel of haemopexin.14

Moreover, an additional Zn2þ-binding site hasbeen reported for PEX2, which is located betweenthe second and third strands of blade IV.11 There-fore, we performed an X-ray absorption scan ofthe PEX9 crystals to detect calcium and othermetal ions bound to PEX9. Intriguingly, this X-rayabsorption scan revealed that neither calcium norzinc is bound in the PEX9 crystals, in contrast tothe previous structural analyses of PEX domains.

Sodium binds to the entrance to the channelof molecule A, but not molecule B

We observe electron density exactly at theposition of the conserved metal ion at the bottomentrance to the propeller channel. It forms contactswith the carbonyl oxygen atoms of Asp522 (bladeI), Asp568 (blade II), and His662 (blade IV) atdistances of 2.56 A, 2.44 A, and 2.36 A, respec-tively (Figure 5). The short distances indicate thatthe solvent molecule is a metal ion rather than awater molecule. On the basis of the X-ray absorp-tion scan, we can rule out the possibility that thisdensity is Ca2þ, as it was interpreted in the PEX1,PEX2 and PEX13 structures.9,11 – 13 Instead, we inter-preted the density at the bottom of the propellerchannel of molecule A as a sodium ion. Thisassignment is consistent with that of the structuresof haemopexin.14,15 In molecule B of the samecrystal, the corresponding density is significantlyweaker and the distances to the above-mentionedligands are 2.68 A, 2.58 A, and 2.79 A. Conse-quently, the solvent at the bottom of the channelof molecule B was interpreted as a water molecule.Blade III contributes neither to metal binding (inmolecule A) nor to water binding (in molecule B).In molecule A, we observed a solvent molecule at2.58 A distance from the sodium ion. Positionedbetween blades I and IV, this water molecule,Wat194, serves as an additional ligand to the Naþ

and is and stabilised further by the carbonyloxygen atom of Phe521 (blade I) (Figure 5).

The lack of Naþ coordination by blade III isrelated directly to the flexibility inherent to theglycine hinge connecting blades II and III. Glycineat position 615 is found exclusively in MMP9(Figure 4) and has a backbone conformationðf;cÞ ¼ ð163:18;2175:98Þ and (166.98, 175.38) in

Figure 3. (a) Detailed view at the PEX9(A)–PEX9(B)dimer interface. Blades IV of PEX9(A) and PEX9(B) arehighlighted in green and red, respectively. Residuesinvolved in more than three interactions are shown asbold sticks. (b) Model of the PEX9(A)–PEX1(B) dimerinterface. The model was obtained by superimposingthe PEX1 structure onto the PEX9(B) structure, asdescribed in the text. In addition to the hydrophobic con-tacts, the Figure shows the two conserved charged inter-actions present in the PEX9(A)–PEX1(B) interface.


Table 1. Interaction matrix of PEX9(A)–PEX9(B)

A

B D651 p655 G656 V657 L659 r677 F678 W680 r685 Q690 V691 V694 g695 Y696 Y699 E706 D707

D651 £P655 £G656 £ £v657l659R677 £ WF678 £W680 £R685 £ £ £Q690 £V691 £V694 £ £ £G695 £Y696 £ £ £ £Y699 £e706d707

Hydrophobic interactions are indicated by the symbol £ , the polar interaction by W. Residues shown with small italic letters are involved in the contact by the alternative molecule only and,thus, highlight the asymmetry of the complex.

molecule A and B, respectively. These confor-mations are energetically unfavourable for anyamino acid residue other than glycine. The Gly615loop deviates between molecule A and B, as indi-cated by the dihedral angles given above, reflectingthe flexibility inherent to the glycine loop. As aconsequence, the relative arrangement of thepropeller blades is significantly more flexible thanin propeller architectures where all four blades arecrosslinked by central metal ions.

b-Propeller blades are not crosslinked byinternal metal ions

Aside from PEX1, in all previously determinedstructures, a minimum of three conserved internalmetal ions chelate all four blades and providerigidity to the propeller architecture. In PEX1, asingle Ca2þ binds all four blades of the b-propellerwhile the other channel-contained solvent mole-cule appear to be water molecules, based on thedistances to their carbonyl ligands.9 In the PEX9structure, however, the internal metal ions are not

conserved. Only molecule A contains a sodiumion at the bottom of the propeller channel, whereit crosslinks blades I, II, and IV, but not blade III.Given the observed repositioning of blades III andIV in PEX9 (Table 2), it becomes evident that thesemetal ions serve a key role in providing stabilityand rigidity to the propeller structure by chelatingall four propeller blades.

Asp663 widens the PEX9 propeller channeland excludes metal ions binding to it

Asp663 marks a particular difference in thestructure of PEX9 channel when compared withall other structures. Asp663 is unique in bindinginto the propeller channel of PEX9. This asparticacid residue is strictly conserved within MMP9throughout different organisms, but with theexception of MMP17 and MMP25, it is not foundin other known MMP sequences (Figure 4).Asp663 contributes to the widening of the channeland thereby displaces the possible metal ligands(carbonyl groups) from the positions observed in

Figure 4. Sequence alignment of selected elements of C-terminal MMP domains from different organisms. Residuescontributing to the PEX9–PEX9 or PEX9–PEX1 contact are highlighted with yellow colour. The charged interaction ofR677 (PEX9B) with the C terminus of PEX9(A) is highlighted in blue and specific to the homodimeric PEX9 complex.Gly615 and Asp663 contribute to the widening of the PEX9 propeller channel.


related structures. Asp663 thus prevents metal ionsfrom binding into the channel (Figure 5). Instead,water molecules are positioned in the channel andestablish an extended hydrogen bonding networkwith the internal strands lining the propellerchannel (Figure 5). The hydrogen bondingdistances are close to 2.8 A.

Strands 1–3 form the core of thepropeller structure

In addition to the above-mentioned widening ofthe channel and repositioning of blades III and IV,

there are a number of additional deviations fromthe known PEX structures, including loop inser-tions within and between the propeller blades.Only strands 1–3 within each blade are topo-logically conserved and can be superimposed. Theouter strand 4 of each blade deviates considerablybetween different PEX domains. This demonstratesthat the inner three strands constitute the struc-tural framework of the b-propeller architecture,while the outer strand of each blade mediatescontacts with other protein components. Similarly,loop insertions between the inner strands conferspecific molecular recognition sites at the bottomand top face of the propeller without disturbingtheir conserved blade architecture.

Electrostatic potential of the monomeric anddimeric PEX9 differ qualitatively

PEX9 exhibits a slightly positive surface chargedistribution, as shown in Figure 6(b). The chargeof the monomeric PEX9 residues appears to bespread quite uniformly over the complete surfacewith absolute minimum and maximum potentialvalues of (260, 83) kBT/e. At its side orientation,PEX9 displays a more neutral potential surface.This includes the surface patch on blade IVforming the predominantly hydrophobic dimerinterface. Notably, the electrostatic potential of thePEX9 dimer differs qualitatively from that of themonomeric molecule (Figure 6(a)). This differenceis reflected also in a change of overall electrostaticproperties, having minimal and maximal potentialvalues of (265, 64) kBT/e. The highly negative sur-face patch is close to the interface of the PEX9dimer and the C termini of both propellers. This isthe site where the Ta6Br12 cluster was bound.

The overall charge of PEX13 is nearly balanced,corresponding to potential values of (271, 74)kBT/e, but significantly clustered. Its top face con-tains a negative groove, whereas positive residuescluster along an extended patch of its bottom face.PEX1 and PEX2 appear to have an overall balancedcharge distribution on their top and bottom face(Figure 6(b)). However, only PEX1 has an approxi-mately neutral charge, with potential values of(271, 82) kBT/e. By contrast, PEX2 is strongly posi-tively charged, with potential values of (253, 99)kBT/e. This positive charge is localised to side

Figure 5. The inner strands forming the channel of thePEX9 propeller are superimposed with those of the PEX2structure (shown in black). The three metal ions usuallyconserved within the channel are indicated as blackballs. The arrows indicate that blade III (yellow) andblade IV (red) of PEX9 are shifted with respect to thePEX2 propeller for more than 1 A. The ligands to theNaþ (carbonyl groups of Asp522, Asp568, His662, andWat194) are shown with the final electron density over-laid. Similarly, the side-chain of Asp663 and the waterstructure inside the channel is shown with the densitycontoured at 1.0s over the mean. The Figure wasprepared by using the programs BOBSCRIPT45 andRaster3D.44

Table 2. Relative shift of the propeller blades of PEX9 compared to related structures

Blade I Blade II Blade III Blade IV

DX DY DZ DX DY DZ DX DY DZ DX DY DZ

PEX9–PEX2 þ0.2 20.8 0.0 þ0.1 20.2 þ0.2 þ0.2 þ0.3 þ1.2 20.3 þ0.1 21.3PEX9–PEX13 þ0.2 20.8 0.0 0.0 0.0 0.0 þ0.2 þ0.5 þ1.2 20.4 20.2 20.8PEX9–hema 20.3 21.3 20.2 þ0.6 20.0 þ0.5 þ0.2 þ0.5 þ1.2 þ0.2 20.3 21.5PEX2–PEX13 0.0 0.0 0.0 20.1 þ0.2 þ0.2 þ0.0 þ0.1 þ0.1 20.1 20.3 þ0.5PEX2–hema 20.5 20.6 20.2 þ0.6 þ0.2 þ0.3 þ0.0 þ0.1 þ0.1 þ0.4 20.4 20.2

The signs of the shifts indicate their direction in A units.a Haemopexin structure reported by Paoli et al.15


edges of the PEX2 propeller. The difference inphysico-chemical properties of PEX9, and theother PEX molecules, translates functionally into aunique affinity profile towards its interactingcomponents.

The whole is greater than the sum of its parts:dimerisation generates newfunctional properties

The dimeric PEX9 features functions that are notpresent in monomeric PEX9. The electrostaticpotential illustrates, at the physico-chemical level,how novel functions are generated by the complex

formation, as described above (Figure 6(a) and(b)). The binding of the Ta6Br12 cluster to thedimeric PEX9 exemplifies how these physico-chemical changes, induced by the dimerisation,may translate into new functional properties.Ta6Br12 binds to the dimer interface by formingcharged interactions with residues Asp707,Tyr699, and Tyr696 (monomer A) as well asAsp706 and Tyr699 (monomer B). The chelation ofthis cluster reflects the asymmetry of the dimerinterface. Moreover, an extended hydrophobic sur-face patch accessible to solvent in the monomericPEX9 becomes buried upon dimerisation. Finally,the dimeric complex brings all domains, including

Figure 6. (a) Electrostatic potential of the homodimeric PEX9 structure. The orientation of the first and second sur-face corresponds to that in Figure 2(a) and (b). The red surface patch denotes the negatively charged reactive site,where the Ta6Br12 cluster was bound. The Figure was prepared by using the programs GRASP,46 MOLSCRIPT43 andRaster3D.44 (b) Panel of solid surface representations of related haemopexin domains, colour-coded by their electro-static potential. The view is along the propeller axis from both the top and bottom orientation. In addition, the surfacerepresentation indicates a wider entrance to the channel from the top side of PEX9 as compared with the other struc-tures. The charge distribution of the monomeric PEX9 is spread rather uniformly, with positive and negative chargesover the complete surface. This contrasts the clustered charge distribution of the dimeric PEX9, where the negativecharge is localised to the Ta6Br12 binding site and positive charges to the opposite site. The Figure was prepared byusing the programs GRASP,46 MOLSCRIPT43 and Raster3D.44


the catalytic domains, of the two gelatinases B towithin a defined distance from each other. Thisdistance restraint is likely to influence a multitudeof functions, including its accessibility for proteo-lytic activation,18 its localisation to the extracellularmatrix,20 and its substrate recognition andprocessing.21

Sulphate-binding sites

Four sulphate groups were bound to the PEX9dimer and are shown in Figure 2(a) and (b)). Thebinding sites are positioned on blades I and III.On the bottom side of blade I, the binding site isformed by the amide groups of Asn519, Ile520 andNz of Lys535. On the top side of blade III, thesulphate-binding site is formed by the side-chainsof Arg618 and Arg621. However, the residuesinvolved in the blade III binding site are notconserved within MMP9. Moreover, this side isoccupied only weakly in molecule B. By contrast,the blade I sulphate-binding site is well defined inboth molecule A and B, and Lys535 is strictlyconserved within the complete MMP family. There-fore, only the sulphate-binding site on blade I, butnot that on blade IV, might be of physiologicalrelevance, possibly in recognition of sulphatedextracellular matrix (ECM) components.

The tetrameric PEX92–TIMP12 cannot beobserved by gel-filtration chromatography

We analysed equimolar PEX9–TIMP1 mixturesby gel-filtration chromatography and SDS-PAGE.We observed a broad peak containing both homo-dimeric PEX92 and PEX9–TIMP1 complexes.However, we could not observe protein eluting atvolumes corresponding to the tetrameric PEX92 –TIMP12 complex. The absence of the tetramericPEX92–TIMP12 complex is another facet under-lining the fact that the monomeric PEX9 anddimeric PEX92 molecules differ significantly.

The heterodimeric PEX9–PEX1 complex

Pro-MMP9 is known to form complexes eitherwith TIMP1, (pro-)MMP1, or (pro-)MMP9.19 All of

these complexes are exclusive, suggesting thatthese complexes involve overlapping recognitionsites. All of the available evidence suggests thatthese complexes involve the C-terminal haemo-pexin domains of MMP1 and MMP9.16,19,22 – 24 Weinvestigated whether the PEX domain of MMP1(PEX1) can dimerise with PEX9 analogously asthe PEX9 homodimer. As pointed out before, thePEX92 homodimer is asymmetric. Therefore, wehad to consider the superposition of PEX1 withmolecule A of the PEX9 dimer, denoted asPEX1(A)–PEX9(B), and with molecule B, denotedas PEX9(A)–PEX1(B). If the PEX1–PEX9 complexmimics the PEX9 homodimer, then there shouldbe a clear preference for one of the two alternativecomplexes. The contact area of these alternativecomplexes provides an objective measure for theirevaluation. The contact area of the PEX9 homo-dimer amounts to 665 A2. The contact area of thecomplex PEX1(A)–PEX9(B) is 470 A2 (correspond-ing to 71% of the PEX92 interface area), while thealternative contact area is 580 A2 (correspondingto 87% of the PEX92 interface area). Consequently,we consider only the latter complex, PEX9(A)–PEX1(B). The corresponding modelled interface isshown in Figure 3(b). Table 3 shows the detailedinteraction matrix of this heterodimeric complex.In particular, there are conserved salt-bridges ofArg453(PEX1) with Asp651(PEX9), and of Ser280and Lys281 of PEX1 with the carboxy terminus ofPEX9. Figure 4 confirms that the involved residuesare conserved in both PEX1 and PEX9.

Discussion

Loosening the structural frameworkaccompanies the dimerisation

One remarkable property of the PEX9 structureis its lack of stabilising elements that are presentin all other related PEX structures, in particularthe crosslinkage of the four propeller blades byinternal metal ions. The PEX9 structure is alsounique as it is the first haemopexin structuredetermined in a dimeric state. The conformationalvariability of Gly615 may in fact provide a

Table 3. Interaction matrix of PEX9(A)–PEX1(B)

PEX9

PEX1 D651 G656 V657 L659 Q675 F678 W680 V691 V694 G695 Y696 D707

S280 WK281 WG421 £ £R453 W £ £ £I454 £ £L455 £ £T456 £L457 £ £ £Q458 £

Hydrophobic interactions are indicated by the symbol £ , polar interactions by W.


switching mechanism by which blade III cancontribute to the Naþ coordination. While Gly615provides the conformational flexibility to evadethe Naþ linkage in the dimeric state, it can adopt aconformation where it binds the central Naþ,thereby crosslinking and stabilising all four propel-ler blades. By analogy with all other PEX struc-tures, the latter state should correspond to themonomeric PEX9. In that case, the Naþ site shouldaid as a conformational switch for the monomer–dimer transition, whereby Naþ occupies themonomeric PEX9 with all four blades binding to it.

Among the other solved haemopexin structures,blades III and IV exhibit a higher degree ofmobility, while the shifts of blades I and II arequite moderate and usually less than 0.5 A. Arelation of the mobility of blades IV and III withtheir involvement in dimerisation is very sugges-tive but not proven, as we do not know thestructure of the monomeric PEX9. The greatermobility of blade IV within all PEX structures mayfurther indicate that this blade serves as a site forprotein recognition, such as PEX2–TIMP2,24,25

PEX1–PEX9,19 or PEX14–PEX14.16

Dimerisation motif of PEX9 may be common toother PEX domains

The residues involved in dimerisation of PEX9are conserved through different MMP9 sequences.This conservation profile is not unique to MMP9(Figure 4). For instance, there is only one salt-bridge stabilising the PEX9 dimer. Other residueswith similar steric and electrostatic propertiesmight substitute the hydrophobic contacts contri-buting to the PEX9 dimer interface. Such relatedresidues are indeed present in PEX1 or PEX14(Figure 4). We modelled the complex of PEX9 withPEX1 by using the PEX9(A)–PEX9(B) dimer asa template. Only the corresponding PEX9(A)–PEX1(B) dimer forms favourable contacts. Theasymmetry of the PEX92 complex thus reflectsits additional competence to form heterophiliccomplexes, such as PEX9(A)–PEX1(B). Given thehigh specificity of macromolecular recognition,this multiple high-affinity binding of PEX9 is trulyremarkable. The binding partner molecule inducesthis adaptive protein recognition by PEX9.

Moreover, the dimerisation of MMP14 (MT1-MMP), which is mediated by its PEX domain(PEX14),16 is likely to employ a recognition motifsimilar to that described for the PEX9 complex.The notion of blade IV mediating the proteinrecognition by haemopexin domains is supportedby its high intrinsic mobility, as evidenced by therelatively large shifts of blade IV (Table 2).

Release of the sodium ion from the channel ofPEX9(B) accompanies dimerisation

The binding of Naþ to molecule A only reflectsthe asymmetry induced by the dimer. The crystalcontacts at the bottom face of molecules A and B

are virtually identical. Therefore, the open channelin molecule B must be a consequence of thedimerisation and the resulting conformationalchanges. This can be seen by specific changes ofinteracting residues, for example within thearray of charged residues Arg630-Glu649-Arg652.Glu649 bridges Arg630 and Arg652 in molecule B,but not in molecule A. Instead, Arg630 of moleculeA forms an alternate salt-bridge with Asp660. Bycontrast, the latter residue forms a hydrogen bondwith Gln675 in molecule B. This difference inthe interaction pattern is accompanied by majorconformational differences of the correspondingresidues in both molecules.

No calcium is bound to PEX9

While the X-ray diffraction data indicate thestrength of the scattering atoms, i.e. the approxi-mate number of electrons, they usually do notallow their direct identification. In many circum-stances, the exact nature of a particular X-rayscatterer can be discriminated only by indirectmeasures, such as its ligand sphere. This explainsthe conflicting interpretation of the bound metalions in the PEX2 structure.11,12 To determine theidentity of bound metal ions unambiguously, theX-ray data often need to be supplemented byemploying additional biophysical or biochemicalmethods. Therefore, we performed an X-rayabsorption scan on the PEX9 crystals to provedirectly the identity of the metals present in thecrystal. The scan clearly rules out that Ca2þ orZn2þ were bound. However, all C-terminal PEXdomain structures of MMPs determined so far con-tained at least one calcium ion in its channel.9,12,13,26

In PEX2, the presence of Ca2þ is necessary forbinding extracellular matrix components such asfibronectin and heparin.26 This necessity is presu-mably related to the intact structural architectureof the PEX2 domain, which will depend on thepresence of crosslinking metal ions inside thechannel, in agreement with the PEX2 crystalstructures.11,12 This situation raises the question ofwhether Ca2þ is able to bind to PEX9 in solution.Its wide channel caused by Asp663 appears to beincompatible with Ca2þ binding. Therefore, weexpect PEX9 to be physiologically independentfrom Ca2þ. Besides MMP9, this aspartic acid isfound only in MMP17 and MMP25. This suggeststhat the propeller channels of the PEX domains ofthese enzymes are similarly widened and also lackmetal binding.

The propeller channel is funnel-shaped andopens towards the top face of the disc-shapedb-propeller. This topological property is commonto all four-bladed b-propeller structures. Propellerscontaining five or more blades usually do not showa similar systematic tunnel widening. It is likelythat this feature relates to the crosslinking metalions that are found in four-blade b-propellersexclusively. The funnel shape of the channel mightprovide a way to sequentially load the haemopexin


domain with the crosslinking metal ions from themore open top side of the channel.

Components possibly interacting with PEX9

PEX9 is known to bind TIMP1,24 TIMP322 anda2(IV) specifically.20 Moreover, (pro-)MMP9 formsa heterodimeric complex with MMP1.19 It is likelythat, similar to PEX2,21,27 PEX9 assists in bindingand processing of the triple-helical collagen IVsubstrate. MMP9 interacts with additional ECMcomponents such as heparin, which inhibitsMMP9 activity.28 Since heparin is a heavilysulphated glycosaminoglycan, it is reasonable toassume that it interacts with MMP9 via its sulphategroups. The conserved sulphate-binding site onblade I of PEX9 (Lys535 together with the amidenitrogen atoms of Asn519 and Ile520) is onesuitable candidate site to mediate this interaction.The channel of PEX9 provides a pronounced cavityon the top side of the b-propeller. This cavity mightbe utilised as a binding pocket by one of theseinteracting factors. The suggested binding pocketis negatively charged, as seen in Figure 6(b), andmight, together with the negative surface areapresent in the dimeric PEX9 (Figure 6(a)), serve asan anchor site for basic surface patches of aninteracting protein.

The interaction of PEX9 with TIMP1

PEX9 is known to bind the C-terminal domain ofTIMP1, but not TIMP2, while PEX2 binds TIMP2,but not TIMP1.24 However, there are contradictoryreports on the existence of the tetramericproMMP92–TIMP12 complex.18 Goldberg andco-workers clearly state that dimerisation ofproMMP9 is incompatible with TIMP1 binding

and, vice versa, that the complex of proMMP9-TIMP1 is incompetent to dimerise.19 This report isin agreement with our findings based on size-exclusion chromatography of the PEX9–TIMP1complex. We did not observe an 80 kDa complex,but only a 40 kDa complex. We conclude that thedimerisation of PEX9 prevents binding of TIMP1either competitively, caused by overlappingcontact areas, or allosterically, caused by confor-mational changes to the PEX9 that render itincompetent for TIMP1 binding.

Allosteric regulation is found more commonlyfor multi-domain proteins.29,30 Therefore, weassume that TIMP1 binding may interfere directlywith the PEX92 complex formation. Figure 7 illus-trates how the C-terminal domain of TIMP1 mightbind to parts of the PEX9 surface patches that areinvolved in the PEX9 homo-dimerisation.

Overall and colleagues performed a Lys/Arg toAla mutagenesis scan of PEX2 to map its TIMP2binding sites.25 Based on their findings, they pro-posed blades III and IV of PEX2 to serve as thebinding site of TIMP2. The mapped PEX2 bindinginterface contains Lys547, Arg561, Lys610, andLys617, corresponding to Arg681, Gln690, Arg634,and Ser619 in PEX9. While the latter two residuesare freely accessible within the dimeric PEX9structure, this is not the case with the former tworesidues. In fact, Gln690 participates in the dimerinterface (Table 1) and is, consequently, buried.The findings reported by Overall et al. thereforesupport the model depicted in Figure 7, where theTIMP1 binding sites overlap with the dimerinterface.

Controlling the dimerisation of PEX domainsas a novel therapeutic approach

Dimerisation emerges as an important mecha-nism to regulate the activation and activity ofMMPs16,18 Given the high impact of MMPs, and inparticular of gelatinases A and B, in pharma-ceutically critical disease areas like cancer, thecontrol of the monomer–dimer transition providesan interesting and welcome novel approach tothe regulation of MMP activity. This approachpromises high specificity by binding the appro-priate PEX (homo- or hetero-) complex of the targetMMP. So far, active site-directed MMP inhibitorshave failed to obtain the desired target specificity.The complex of Ta6Br12 with the PEX9 dimerprovides an example of how low molecular masscompounds can be utilised to either stabilise orinterfere with the PEX9 complex. On the basis ofthe structure presented here, it should be possibleto uncouple the PEX92 homo-dimerisation fromthe PEX9–TIMP1 complex formation by using alow molecular mass compound that, for example,prevents the PEX92 dimerisation but not thePEX9–TIMP1 complex. Such a compound wouldrender MMP9 inhibited more tightly by TIMP1,which is desirable in several cancers.31,32

Figure 7. A representation of the overlapping contactsinvolved in the PEX9 homodimer and PEX9–TIMP1heterodimer. The model reflects the fact that the C-termi-nal domain of TIMP1 binds PEX9.47,48 The Figure wasprepared by using the programs GRASP,46 MOLSCRIPT43

and Raster3D.44


Table 4. Data collection and phasing statistics

CompoundWavelength

(A)Resolution

(A) Observations Unique reflectionsCompleteness

(%) Rmerge £ 100a Riso £ 100b SitesPhasing power(a/c) at 4.5 Ac

Rcullis

(a/c/ano) at 4.5 Ad

Native 1.0500 20–1.95 124,981 36,949 97.8 8.2/48.8 – – – –Ta6Br12 (Ta peak) 1.2548 20–2.6 75,616 29,389 98.6 4.8/28.8 22.3/28.5 1 2.44/1.33 0.54/0.67/0.69Ta6Br12 (Ta inflect.) 1.2554 20–2.6 74,955 29,336 98.5 4.9/29.0 22.1/27.6 1 2.37/1.28 0.55/0.68/0.74Ta6Br12 (Ta remote) 1.2300 20–2.6 40,179 16,401 97.5 4.8/35.7 23.1/29.7 1 2.43/1.38 0.54/0.64

a Rmerge ¼P

i

Ph lIðhÞi 2 kIðhÞll=

Ph IðhÞ; where I(h)i is the ith measurement of the reflection Iðh; k; lÞ and kIðhÞl is the mean intensity for that reflection. The two values indicate the full resolution

range and the outer resolution shell.b Riso ¼

PlFPH 2 FPl=

PFP; where FPH and FP refer to the derivative and native structure factor, respectively. The two values again indicate the full resolution range and the outer resolution shell.

c Phasing power (acentric/centric) ¼ klFHll=klFPH 2 lFP þ FHlll; where FH denotes the heavy atom structure factor; the denominator is also known as residual lack of closure (E).d Rcullis (acentric/centric/anomalous) ¼ klFPH 2 lFP þ FHlll=klFPH 2 FPll:

Materials and Methods

Cloning and protein expression

All DNA manipulations were performed usingstandard techniques.33 The coding gene of PEX9 wasamplified from a human prostate cDNA library(Clontech 1131a) by using a nested PCR method. Theinitial primers for the forward and reverse directionread GTG AAC ATC TTC GAC GCC and CCT ATGACA TCC TGC AGT G, respectively. In the second PCRstep SphI (forward) and CelII (reverse) restriction siteswere introduced. This PCR product was subcloned intoa pDS expression vector under the control of a T5promoter†. DNA sequencing of both directions of thegene, including promoter and terminator region con-firmed the correctness of the plasmid. The coding genefor TIMP1 was prepared by total synthesis, whichallowed optimisation of the codon usage for the E. colihost. Both proteins were expressed in E. coli K12 strainUT560034 carrying the LacIq repressor plasmid pUBS-520.35 Fermentation was performed in a 10 l scale at37 8C and expression was induced with 1 mM IPTG atan A595 nm of 0.8–1.0.

Folding and purification

The cells were harvested by centrifugation andopened by French press. The inclusion body proteinwas solubilised in 6 M guanidinium–HCl, 20 mMEDTA, 150 mM oxidised L-glutathione (GSSG), 15 mMreduced glutathione (GSH) and 100 mM Tris–HCl (pH8.5), and then dialysed against water slightly buffered topH 4.7 at 4 8C. After centrifugation, the pellet wasdissolved in 55% guanidinium–HCl (pH 4.5). The dis-solved protein was diluted rapidly in refolding buffer(0.8 M L-arginine, 0.1 M NaCl, 10 mM CaCl2, 1 mMZnCl2, 50 mM Caps (pH 10.0), 0.5 mM cysteine mono-hydrate) and left for three days at 16 8C. For TIMP1, thepH of the refolding buffer was adjusted to 7.5, whichreduces the rate of disulphide bond formation. Toremove the arginine, the refolded protein was dialysedagainst 25 mM Caps (pH 10.0), 10 mM CaCl2, 100 mMNaCl, 1 mM ZnCl2. After separating precipitated proteinby centrifugation, the soluble refolded protein was con-centrated. The pH of both proteins was adjusted to 7.0with HCl. In a first purification step, either protein wasloaded onto an SP-Sepharose column, which was equili-brated with 25 mM Tris–HCl (pH 7.0), 20 mM arginine.PEX9 eluted at about 450 mM NaCl, TIMP1 at about400 mM NaCl. For final purification, either protein wassubjected to gel-filtration using a Superdex75 columnwith a running buffer of 10 mM Tris–HCl (pH 7.0),250 mM NaCl, 20 mM arginine.

Crystallisation, data collection and processing

PEX9 was crystallised by using the vapour-diffusionmethod in a sitting drop. The protein (1 ml at a concen-tration 10 mg/ml) was mixed with 1 ml of the reservoirsolution (100 mM Tris–HCl (pH 7.5), 30% (w/v) poly-ethylene glycol monomethyl ether (PEGMME) 5000,200 mM ammonium sulphate). Crystals appeared afterfive days at room temperature and grew to maximum

dimensions of 0.1 mm £ 0.1 mm £ 2 mm. Crystals wereflash-frozen in a cold nitrogen stream. High-resolutiondata were collected at the synchrotron beamline BW6 atDESY Hamburg. For the tantalum derivative data collec-tion, crystals were soaked with Ta6Br12 for one week,after which the crystals were deeply green coloured. Athree-wavelength, multiple anomalous dispersion(MAD) experiment was performed at the tantalum LIIIabsorption edge (Table 4). Data were processed usingthe programs DENZO36 and MOSFLM.37 The data werescaled with programs of the CCP4 package.38 The deriva-tive data were re-indexed to match the unit cell setting ofthe native data.

Structure determination

Conventional as well as exhaustive six-dimensionalPatterson searches failed to localise homologous searchmodels (PEX2, PEX13, PEX1) in the PEX9 crystal. Retro-spectively, this failure can be ascribed, at least partly, tothe propeller blade rearrangement due to the missingcrosslinkage by internal ions. The tantalum site wasinterpreted by anomalous and isomorphous differencePatterson techniques.39 Initial attempts to resolve theTa6Br12 cluster failed, although the cluster adopts a well--defined orientation, as could be shown by using the finalphase set. Therefore, single isomorphous replacement(SIR)-MAD phasing was limited to 4.5 A resolution.38

The solvent-flattened experimental density revealedclear solvent boundaries. The electron density was cutout using the program MAIN.40 The rotation matrixrelating the two molecules was determined in reciprocalspace by calculating the rotation function of the Fourier-transformed molecular densities.41 The translational partof the non-crystallographic symmetry (NCS) operatorwas determined by real space correlation scans.40 Usingthe derived NCS operators, phases were extended byNCS averaging to 2.0 A.40 The resulting map easilyallowed tracing the model in the electron density usingMAIN. NCS restraints were released at the final steps ofmodel refinement42 to account for the deviation fromthe 2-fold local symmetry.

Model refinement was performed using the programCNS,42 water molecules were picked automatically inthe difference density using the appropriate routine ofCNS and verified manually. The final dimeric modelcontains residues Asp513 to Asp707 in both moleculesof the dimer, 200 water molecules, four sulphate ionsand one Naþ. The refinement statistics of the currentmodel is summarised in Table 5.

Table 5. Refinement statistics

Refinement

Rcryst (%) 22.9Rfree (%) 27.5r.m.s. deviation in bond lengths (A) 0.008r.m.s. deviation in bond angles (deg.) 1.36r.m.s. bonded B (A2) 1.6Average B-factor (A2) 27.5Space group P43

Unit cell dimensionsa, b, c (A) 127.72, 127.72, 31.38

† Kopetzki, E., Rudolph, R. & Grossmann, A. (1993).Recombinant core streptavidin. Boehringer Mannheim,USA.


Protein Data Bank accession code

The coordinates and structure factors have beendeposited with the RCSB Protein Data Bank under thePDB entry code 1ITV.

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Edited by D. Rees

(Received 18 February 2002; received in revised form 15 May 2002; accepted 22 May 2002)


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Structural Basis of the Adaptive Molecular Recognition by MMP9