Dynamic Interdomain Interactions Contribute to theInhibition of Matrix Metalloproteinases by Tissue Inhibitorsof Metalloproteinases*□S

Received for publication, November 3, 2010, and in revised form, April 14, 2011 Published, JBC Papers in Press, April 25, 2011, DOI 10.1074/jbc.M110.200139

Albert G. Remacle, Sergey A. Shiryaev, Ilian A. Radichev, Dmitri V. Rozanov, Boguslaw Stec, and Alex Y. Strongin1

From the Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037

Because of their important function, matrix metalloprotei-nases (MMPs) are promising drug targets in multiple diseases,including malignancies. The structure of MMPs includes a cat-alytic domain, a hinge, and a hemopexin domain (PEX), whichare followed by a transmembrane and cytoplasmic tail domainsor by a glycosylphosphatidylinositol linker in membrane-typeMMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent naturalregulators of the MMP activity. These are the inhibitory N-ter-minal and the non-inhibitory C-terminal structural domains inTIMPs. Based on our structuralmodeling, we hypothesized thatsteric clashes exist between the non-inhibitory C-terminaldomain of TIMPs and the PEX of MMPs. Conversely, a certainmobility of the PEX relative to the catalytic domain is requiredto avoid these obstacles. Because of its exceedingly poorassociation constant and, in contrast with TIMP-2, TIMP-1 isinefficient against MT1-MMP. We specifically selected anMT1-MMP�TIMP-1 pair to test our hypothesis, because anyimprovement of the inhibitory potency would be readilyrecorded. We characterized the domain-swapped MT1-MMPchimeras in which the PEX of MMP-2 (that forms a complexwith TIMP-2) and of MMP-9 (that forms a complex withTIMP-1) replaced the original PEX in theMT1-MMP structure.In contrast with the wild-type MT1-MMP, the diverse proteo-lytic activities of the swapped-PEX chimeras were then inhib-ited by both TIMP-1 and TIMP-2. Overall, our studies suggestthat the structural parameters of bothdomains ofTIMPshave tobe taken into account for their re-engineering to harness thetherapeutic in vivo potential of the novel TIMP-based MMPantagonists with constrained selectivity.

There are 24 individual MMPs2 in humans. MMPs cleavemultiple extracellular matrix components, growth factors,

cytokines, and cell signaling adhesion receptors. Aberrant per-formance ofMMPs plays a role in a plethora of diseases, includ-ing cancer (1–3). There are 18 soluble and 6membraneMMPs.The structure of soluble MMPs includes a prodomain (PRO), acatalytic domain (CAT) that contains the active site zinc, ahinge, and a PEX. Additionally, membrane-type MMPs (MT-MMP) contain a transmembrane domain followed by a shortcytoplasmic tail (CYTO) (MT1-MMP, MT2-MMP, MT3-MMP, andMT5-MMP) or a glycosylphosphatidylinositol moi-ety (MT4-MMP and MT6-MMP) that anchors the proteinaseto the cell surface (1, 4, 5).MMPs are synthesized as zymogens, which require the pro-

teolytic processing of the N-terminal inhibitory PRO to gener-ate the active enzymes (4). It is accepted that secretory tissueinhibitors of MMPs (TIMPs) play an important role in the reg-ulation of the proteolytic activity of MMPs (6, 7). Four TIMPs(TIMP-1, -2, -3, and -4) are present in humans (8). There is atleast a 25% sequence identity among all TIMPs, including 12conserved Cys residues that form 6 disulfide bridges resultingin 6 loop regions. There are two domains, N-terminal andC-terminal, in TIMPs. An N-terminal domain (NT-TIMP)binds the CAT, carries the MMP-inhibitory activity, andencompasses the three first loops. A non-inhibitory C-terminaldomain (CT-TIMP) binds, albeit with distinct affinities, thePEX of MMPs. TIMP-1 forms a stoichiometric complex withtheMMP-9 proenzyme via the binding of its CT-TIMPwith thePEX (9). In turn, TIMP-2 forms a complex with the MMP-2proenzyme (10, 11).Truncated NT-TIMP folds correctly and exhibits a signifi-

cant level of the MMP-inhibitory activity. Crystal structures ofmultiple TIMP�MMP inhibitory complexes have been deter-mined. They demonstrate similar inhibitory mechanisms,involving a region surrounding the conserved Cys1–Cys70

disulfide bond of TIMPs and especially the N-terminal Cys1–Pro6 and Glu67–Cys70 segments (numbering is given accordingto TIMP-1) that approach the MMP active site (PDB 1UEA,1BUV, 1GXD, 1BR9, and 2E2D) (12–18). TheCys1 coordinates,via bidentate interactions, the catalytic zinc of the MMP activesite.Normally, TIMPs performas nanomolar or even femtomo-lar range inhibitors of MMPs (8, 19–22).Multiple studies have been performed to constrain the spec-

ificity of TIMPs and transformTIMPs into selective rather thanwide-ranging inhibitors (14, 23–26). The inhibitory NT-TIMPalone and the individual CAT of MMPs were predominantlyused in these studies. Differences in contacts and chemistry ofthe interfaces of TIMP�MMP complexes provide a basis for re-

* This work was supported, in whole or in part, by National Institutes of HealthGrants CA77470 and CA83017 (to A. Y. S.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1–3.

1 To whom correspondence should be addressed: Cancer Research Center,Sanford-Burnham Medical Research Institute, La Jolla, CA 92037. Tel.: 858-795-5271; Fax: 858-795-5225; E-mail: strongin@sanfordburnham.org.

2 The abbreviations used are: MMP, matrix metalloproteinase; CAT, catalyticdomain; CT-TIMP, C-terminal TIMP domain; NT-TIMP, N-terminal TIMPdomain; EZ-Link sulfo-NHS-LC-biotin, sulfosuccinimidyl-6-(biotinamido)-hexanoate; EZ-link sulfo-NHS-SS-biotin, sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate; Mca-PLGL-Dpa-AR-NH2, methoxycoumarin-4-yl-acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl)-Ala-Arg-NH2; MESNA, 2-mercaptoethane sulfonic acid; MT1-MMP, membranetype-1 MMP; PEX, hemopexin domain; PRO, prodomain; TIMP-1 and -2, tissueinhibitor of MMPs-1 and -2.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 23, pp. 21002–21012, June 10, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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engineering of the inhibitory selectivity. Because of its exceed-ingly poor association constant, TIMP-1 is incapable of effi-ciently inhibiting the full-length MT1-MMP enzyme, albeit acertain level of inhibition was observed with the individual cat-alytic domain of MT1-MMP (21, 23, 24). A single point muta-tion of Thr98 to Leu (T98L) at a distal site from the inhibitoryloop, however, increased the association constant and trans-formed the NT-TIMP of TIMP-1 into a tight-binding inhibitorof MT1-MMP (14, 27).There are, however, additional important structural ele-

ments, which are outside of the inhibitory and catalyticdomains of the inhibitor and the proteinase, respectively. Thus,the contacts of the CT-TIMP of TIMP-1 and TIMP-3 arerequired with the C-terminal, non-catalytic domain to achievean efficient inhibition of a disintegrin and metalloprotease-10(ADAM10). The NT-TIMP alone is insufficient for the inhibi-tion of ADAM10 (28). Conversely, the full-length TIMP-4 is aweek inhibitor of ADAM17 (tumor necrosis factor-alpha con-verting enzyme; TACE), whereas the C-terminal truncationsignificantly increases the inhibitor’s potency (29). Overall,both the CT-TIMP and its counterpart, the C-terminal non-catalytic PEX in MMPs, play a likely important, albeit uniden-tified, role in themechanisms of inhibition ofMMPs by TIMPs.To highlight the effect of the PEX on the interactions of

MT1-MMPwith the full-length TIMPs, we designed and char-acterized the MT1-MMPmutants with the truncated domainsand also the MT1-MMP chimeras. In these chimeras the orig-inal PEX was substituted in the MT1-MMP molecule by thePEX derived from MMP-2 and MMP-9. We specificallyselected MMP-2 and MMP-9 because of their contrastingproenzyme complex formations with TIMP-2 and TIMP-1,respectively. In addition, we specifically selected TIMP-1 forour experiments because of its exceedingly low ability to inter-act withMT1-MMPand because any improvement of the affin-ity of TIMP-1 would be readily recorded. Based on our experi-ments, we now suggest that the interactions between the PEXofthe full-lengthMT1-MMPand theCT-TIMPalso contribute tothe inhibitory efficacy of TIMPs. As it appears now, the globalfold of the PEX, the interdomain dynamics, and the intrinsicprotein flexibility of bothMMPs andTIMPs play a role in defin-ing the binding interface in the course of inhibition of the full-length cellularMMPs by the full-length TIMPs. From the prac-tical perspectives, it is likely that constraining the specificity ofTIMPs toward the individual full-length MMPs also requiresthe modification in the non-inhibitory CT-TIMP and that themodifications in the inhibitory NT-TIMP alone are insufficientfor the re-design of the inhibitor specificity.

MATERIALS AND METHODS

General Reagents and Antibodies—All reagents are pur-chased from Sigma-Aldrich unless indicated otherwise. Amurine monoclonal antibody against the CAT of MT1-MMP(clone 3G4) and a hydroxamate inhibitor of MMPs (GM6001)were purchased from Chemicon. A SuperSignal West DuraExtended Duration Substrate kit, EZ-Link sulfo-NHS-SS-bio-tin (sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropi-onate), and EZ-Link sulfo-NHS-LC-biotin (sulfosuccinimidyl-6-(biotinamido)hexanoate) were from Pierce. The secondary

species-specific antibodies conjugated with HRP were pur-chased from Jackson ImmunoResearch. The secondary species-specific antibodies conjugated with Alexa Fluor 594 (red) wereobtained from Molecular Probes. A proteinase inhibitor mix-ture set (Set III) was from Calbiochem.Recombinant Proteins—Recombinant human TIMP-1 was

obtained from Invitrogen. Recombinant TIMP-2 was purifiedfrom the medium conditioned by the recombinant CHO cells(30). The secretory N-end-appended TIMP-2 mutant(A-TIMP-2) that exhibited the Glu-Ala-Glu-Ala-Tyr-Val-Glu-Phe sequence attached to the N-terminal Cys1 was purifiedfrom the recombinant Pichia pastoris yeasts using FPLC on aMono-Q column (31). The TIMP-2-free MMP-2 proenzymewas isolated from p2AHT2A72 cells derived from the fibrosar-coma HT1080 cell line sequentially transfected with the E1Aand MMP-2 cDNAs (32). The individual CAT of MT1-MMPand MT6-MMP was expressed in Escherichia coli, purifiedfrom the inclusion bodies, and refolded to restore its catalyticactivity (33, 34). The concentrations of the purified, catalyti-cally active MT1-MMP and MT6-MMP were measured bytitration against a standard GM6001 solution of a known con-centration and Mca-PLGL-Dpa-AR-NH2 as a fluorescent pep-tide substrate (35).Cloning of theMT1-MMPChimeras—Todesign both chime-

ras, we used the cDNA coding for the full-length human wild-type MT1-MMP (WT), MMP-2, and MMP-9 cDNAs. Thesequences coding for the PEX of MMP-2-(470–659) andMMP-9-(517–703) were each inserted between Cys319 andCys508 of the MT1-MMP sequence to generate the PEX/MMP-2 and PEX/MMP-9 chimeras, respectively. The authen-ticity of the recombinant constructs was confirmed by DNAsequencing.Cells—Human breast carcinoma MCF-7 cells were obtained

from ATCC (Manassas, VA). The full-length human �3 integ-rin subunit and MT1-MMP cDNAs were each cloned into thepcDNA3.1-neo and pcDNA3.1-zeo vectors, respectively (Invit-rogen) (36). The catalytically inactive MT1-MMP mutant(E240A) was generated by replacing the essential Glu240 of theproteinase active sitewithAla (37). TheC-end-truncatedMT1-MMP (�CYTO) mutant lacking the 563–582 CYTO sequencewas described earlier (38). The inert mutant lacking the 112–284 sequence of the CAT (�CAT) and the mutant lacking the319–508 sequence of the PEX (�PEX) were described earlier(39). Cell transfections were performed using Lipofectamine(Invitrogen) according to the manufacturer’s instructions. Togenerate double transfectants, the parental MCF-7 cells werefirst transfected with the full-length �3 integrin chain cDNA inthe pcDNA3.1-neo plasmid (MCF7-�3 cells) as described ear-lier (40). MCF7-�3 cells were next transfected with the originalpcDNA3.1-zeo vector or the pcDNA3.1-zeo plasmid encodingfor theWT, E240A, �CAT, �PEX, �CYTO, PEX/MMP-2, andPEX/MMP-9 constructs to generate the respective stably trans-fected cell lines. Routinely, transfected cell lines were main-tained in DMEM-10% FBS supplemented with G418 and/orZeocin (0.2 mg/ml each).In addition, to quantitatively assess the inhibitory potency of

TIMP-1 and TIMP-2 against the WT and PEX/MMP-9 con-structs, we employed MCF7-�3 cells transiently transfected

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with the pcDNA3.1-zeo plasmid encoding for WT and PEX/MMP-9. The cells were transfected with the recombinant plas-mids using Lipofectamine LTX and the Plus reagent (Invitro-gen) according to the manufacturer’s protocol. The cells wereused in our inhibitory experiment in 48 h post-transfection.Cell Surface Biotinylation and 2-Mercaptoethane Sulfonic

Acid (MESNA) Treatment—Cells were grown in wells of a6-well plate in the presence of GM6001 (50 �M). Cell were thensurface-biotinylated using non-cleavablemembrane-imperme-able EZ-Link sulfo-NHS-LC-biotin (0.3 mg/ml in Sorensenphosphate buffer, pH 7.8, containing 14.7 mM KH2PO4, 2 mM

Na2HPO4, and 120 mM sorbitol) (41). Cells were next lysedusing 50 mM N-octyl-�-D-glucopyranoside in Tris-bufferedsaline supplemented with 1 mM PMSF, 1 mM CaCl2, 1 mM

MgCl2, 10 mM EDTA, and proteinase inhibitor mixture set III(N-octyl-�-D-glucopyranoside buffer). The biotin-labeledplasma membrane proteins were pooled down using streptavi-din beads (42). The precipitates were dissolved in SDS samplebuffer (0.125 M Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 0.005%Bromphenol Blue) with 100mMDTT and analyzed byWesternblotting with theMT1-MMP antibody followed by the second-ary HRP-conjugated antibody and a SuperSignal West DuraExtended Duration Substrate kit.For the uptake experiments, cells were surface-biotinylated

using the cleavable membrane-impermeable EZ-Link sulfo-NHS-SS-biotin. Immediately following the biotinylation proce-dure, cells were incubated for 25 min at 37 °C in serum-freeDMEM supplemented with 1% insulin-transferrin-selenium toallow the internalization of biotin-labeled MT1-MMP (41, 42).To remove the residual cell surface biotin, cells were incubatedfor 25 min on ice in Sorensen phosphate buffer containingmembrane-impermeable MESNA (150 mM). Cells were nextextensively washed using Sorensen phosphate buffer and lysed,and the lysates were precipitated using streptavidin beads andanalyzed as above.Cell Treatment with MMP Inhibitors—Cells (2 � 105) were

grown in DMEM-10% FBS for 18 h. Where indicated, themedium was replaced with serum-free DMEM containingTIMP-1 (100 nM), TIMP-2 (100 nM), A-TIMP-2 (100 nM), orGM6001 (50 �M). Cells were washed with PBS and lysed in theN-octyl-�-D-glucopyranoside buffer. Insoluble material wasremoved by centrifugation (10,000 � g; 15 min). The superna-tant aliquots (3 �g of total protein) were analyzed under reduc-ing conditions by Western blotting with the MT1-MMP anti-bodies as above.Gelatin Zymography—Cells (2� 105) were seeded for 24 h in

DMEM-10% FBS in wells of a 24-well plate. The medium wasthen replaced with fresh serum-free DMEM (0.225 ml/well)supplemented with pro-MMP-2 (0.5 nM). Where indicated,TIMP-1 (1–1,000 nM), TIMP-2 (1–1,000 nM), A-TIMP-2 (100nM), or GM6001 (50 �M) was added to the samples. In 24 h themedium aliquots (20 �l) were analyzed by gelatin zymographyin 0.1% gelatin-10% acrylamide gels. Alternatively, to concen-trate the samples 3- to 4-fold and, as a result, to increase thesensitivity of the procedure (e.g. with the MT1-MMP chime-ras), the 150-�l medium aliquots were precipitated at 4 °C for16 h using gelatin-Sepharose 4B beads (20 �l of a 50% slurry),

eluted using 50 �l of SDS sample buffer, and a half of the elutedmaterial was analyzed by gelatin zymography.Enzymatic Assay—MMP activity was measured in triplicate

in wells of a 96-well plate in 0.2 ml of 50 mM HEPES, pH 7.5,containing 10 mM CaCl2 and 50 �M ZnCl2. Mca-PLGL-Dpa-AR-NH2 (10 �M) was used as a fluorescent substrate. The con-centration of MT1-MMP and MT6-MMP in the reactions was5 nM. The steady-state rate of substrate hydrolysis was moni-tored continuously (�ex � 320 nm and �em � 400 nm) at 37 °Cfor 3–25 min using a fluorescent spectrophotometer. Whereindicated, TIMP-1 (25–125 nM) and TIMP-2 (25–125 nM) wereco-incubated for 30 min at 20 °C with the MMP samples priorto adding the substrate.Immunostaining of Cells—Cells grown on 15-mm glass cov-

erslips were fixed for 20 min with 4% formaldehyde. Whereindicated, cells were permeabilized for 4 min using 0.1% TritonX-100 or left untreated. Cells were then blocked for 1 h at ambi-ent temperature using 10% BSA in PBS and then stained over-night at 4 °C with the MT1-MMP 3G4 antibody (dilution1:1000) or the polyclonal rabbit MT1-MMP AB815 antibody(dilution 1:200) followed by a 1-h incubation with the second-ary species-specific antibody (dilution 1:200) conjugated withAlexa Fluor 594. The slides were mounted in the Vectashieldmedium containing DAPI for the nuclear staining. The slideswere analyzed using an Olympus BX51 fluorescence micro-scope equipped with a MagnaFire digital camera.In Situ Gelatin Zymography Using FITC-gelatin—FITC-gel-

atin was prepared as described earlier (33). Cells (1� 104) wereseeded onto the gelatin-coated coverslips and incubated for16 h at 37 °C in serum-free DMEM supplementedwith TIMP-1(100 nM), TIMP-2 (100 nM), or GM6001 (50�M). The cells werethen fixed with 4% formaldehyde for 16 min, permeabilized for4 min using 0.1% Triton X-100, and stained for MT1-MMP asdescribed above. The dark regions of degraded FITC-gelatincan be readily detected using a fluorescent microscope.Structural Modeling—The structural coordinates of the por-

cine full-length MMP-1 enzyme complexed with a specificinhibitor N-[3-N�-hydroxycarboxamido)-2-(2-methylpropyl)-propananoyl]-O-methyl-L-tyrosine-N-methylamide (PDB 1FBL)(43), the human MMP-1 E200A mutant proenzyme (PDB1SU3) (44), the human MMP-2 E385A mutant proenzymecomplexed with TIMP-2 (PDB 1GXD) (17), the human full-lengthMMP-2E404Amutant proenzyme (PDB1CK7) (45), theindividual CAT of human MMP-3 in complex with humanmutant (N30A and N77A) TIMP-1 (PDB 1UEA) (13), the indi-vidual CAT of MT1-MMP complexed with bovine TIMP-2(PDB 1BUV) (12), the individual CAT of MT1-MMP com-plexed with the C-terminally truncated, V4A, P6V, and T98Ltriplemutant TIMP-1 (PDB 3MA2) (14, 23), the individual PEXof humanMMP-2 (PDB 1RTG) (46), and the individual PEX ofMT1-MMP (PDB3C7X)were used in our study. The structureswere analyzed and superimposed using PyMOL. The imageswere prepared also in PyMOL.

RESULTS

TIMP�MMP Inhibitory Complex—Like all of the other mem-bers of the MMP family, the active MT1-MMP proteinase isregulated by TIMPs. The association constant of TIMP-1 with

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the MT1-MMP enzyme, however, is exceedingly poor. Thisassociation constant is significantly less efficient comparedwith those of TIMP-2 and TIMP-3 (23, 24). As a result, TIMP-1is not capable of inhibiting the cellular MT1-MMP activity,especially under a physiological range of inhibitor concentra-tions and especially if compared with TIMP-2 (8, 21). Our workand multiple publications by others suggested that the PEX isinvolved in theMMP homodimerization and the association ofthe individual MMPs with TIMPs as well as in the interactionsofMMPswith their cleavage substrates (9, 10, 31, 47–53). Basedon this general assumption, we hypothesized that the structureof the PEX also contributes to the inhibitory interactions ofactive MMPs with TIMPs.To support our hypothesis, we performed a thorough super-

imposition analysis of the availableMMP andTIMP structures,includingPDB1FBL, 1SU3, 1GXD, 1CK7, 1UEA, 1BUV, 3MA2,1RTG, and 3C7X. Thus, the overall fold of the PEX in theMMP-1 proenzyme (PDB 1SU3) is highly similar with that ofthe individual PEX of MMP-2 (PDB 1RTG) and MT1-MMP(PDB 3C7X). The PEX position, however, is shifted relative tothe CAT in the MMP-1 enzyme (PDB 1FBL) compared withtheMMP-1 proenzyme (PDB 1SU3). Similarly, there is a differ-ence in the relative positions of the PEX and the CAT in theMMP-2 proenzyme alone (PDB 1GXD) and the MMP-2proenzyme�TIMP-2 complex (PDB 1CK7). Our estimate sug-gests that the mobility range of the PEX relative to the CATexceeds 10° as measured from the extreme conformationalstates in the MMP-1 (PDB 1FBL and 1SU3) and MMP-2 (PDB1GXD and 1CK7) structures we analyzed (supplemental Fig.S1). These structural differences are distinct and additional tothose local differences that were observed in the interfacebetween the PRO and the PEX (44).In a similar fashion, the overall fold of TIMP-1 andTIMP-2 is

highly similar in their respective complexes withMMP-3 (PDB1UEA) and MT1-MMP (PDB 1BUV and 3MA2). Notably, asignificant difference is in the AB loop of NT-TIMP because ofthe protruding six-amino acid insert in TIMP-2 relative toTIMP-1 (54). There is, however, an�15° difference in the posi-tion ofTIMP-1 andTIMP-2 bound to theCATofMMP-3 (PDB1UEA), and MT1-MMP (PDB 1BUV and 3MA2), respectively(supplemental Fig. S2).Taken together, our analysis suggests that a certain level of

motion of the PEX is required to permit an inhibitoryTIMP�CAT complex. In the absence of this motion, the CT-TIMP collides with the PEX moiety. As a result, the NT-TIMPappears incapable of the productive inhibitory interactionswith the CAT active site (Fig. 1; supplemental Fig. S3). Natu-rally, molecular packing interactions in the crystal may affectthe relative orientation of domains in multi-domain proteins.The mobility of the PEX relative to the CAT domains werecorded, however, significantly exceeds these parameters. Todetermine experimentally if the PEXaffects the inhibitory char-acteristics of TIMPs, we performed an extensivemutagenesis ofthe MT1-MMP sequence followed by a characterization of themutants, including the analysis of their sensitivity to the inhi-bition by TIMP-1 and TIMP-2.Expression and Analysis of the MT1-MMP Constructs—To

identify the role of the individual structural domains in the

functionality of MT1-MMP, we constructed mutants in whichthe CAT, the PEX, and the CYTO were truncated in the MT1-MMP sequence (�CAT, �PEX, and �CYTO, respectively). Wealso constructed theMT1-MMP chimeras in which the PEX ofMMP-2 and MMP-9 replaced the original PEX in the MT1-MMP sequence (PEX/MMP-2 and PEX/MMP-9, respectively).As controls, we used the wild-type MT1-MMP construct andthe E240Amutant in which Ala substituted for the catalyticallyessential Glu240 residue of the proteinase active site (Fig. 2A).

The constructs were then stably co-expressed with the �3integrin subunit in human breast carcinoma MCF-7 cells. We

FIGURE 1. The binding of TIMPs to the CAT modifies the relative positionof the PEX in MMPs. This figure summarizes a detailed superimposition anal-ysis of the available crystal structures of the proenzyme and the enzyme ofMMP-1 (PDB 1SU3 and 1FBL, respectively), of pro-MMP-2 alone and the pro-MMP-2�TIMP-2 complex (PDB 1CK7 and 1GXD, respectively), of the individualCAT of MMP-3�TIMP-1 complex (PDB 1UEA), of the individual CAT of MT1-MMP�TIMP-2 complex (PDB 1BUV), of the individual CAT of MT1-MMP com-plexed with the C-terminally truncated TIMP-1 (PDB 3MA2), and of the indi-vidual PEX of MMP-2 and MT1-MMP (PDB 1RTG and 3C7X, respectively)(supplemental Figs. S1–S3). A, interactions of TIMPs with the CAT and the PEXof active MMPs. After removal of the PRO, active MMPs bind TIMPs by theirCAT in a way that the inhibitory NT-TIMP occupies the space of the removedPRO. Our modeling suggests that the non-inhibitory CT-TIMP interferes withthe PEX. A steric clash between the CT-TIMP and the PEX is indicated by theyellow highlighting. To escape this interdomain clash, the PEX bends awayfrom the CT-TIMP to adopt a new position (the dotted line) that is 10° clock-wise (the red arrow) relative to its original position (the solid line). If this PEXshift does not take place, the productive inhibitory position of the NT-TIMPcannot be achieved. B, stereo view of the molecular surface representation ofthe modeled MMP-1�TIMP-1 and MMP-1�TIMP-2 complexes. Central panels,the structure of the full-length active MMP-1 alone (PDB 1FBL, yellow) wassuperimposed with the structures of the individual CAT of MMP-3�TIMP-1complex (PDB 1UEA, red; only TIMP-1 is shown) and of the individual CAT ofMT1-MMP�TIMP-2 complex (PDB 1BUV, blue; only TIMP-2 is shown). The puta-tive region of the steric clashes between the PEX and the CT-TIMP is boxed.The line indicates the cross-section site. The black arrows indicate the direc-tion of the view in the left and right panels. The left and right panels show a 90°rotated view of the bottom portion of the cross-sections of the MMP-1�TIMP-1and MMP-1�TIMP-2 complexes, respectively. Note the putative penetration ofloop 6 of the CT-TIMP moiety into the space occupied by the PEX.

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specifically selected �3 integrin-transfected MCF-7 cells as thehost for our experiments. Similarly to the parentalMCF-7 cells,�3 integrin-transfected cells express neither MT1-MMP norMMP-2. The �3 integrin-transfected cells, however, exhibithigh levels of the fully functional �V�3 integrin (36, 40). As aresult, �3 integrin-transfected cells are easy to handle com-pared with MCF-7 cell transfected with MT1-MMP alone.To assess the expression level and the catalytic activity of

MT1-MMP, the obtained stably transfected cells were thenexamined by Western blotting and gelatin zymography (Fig.2B). The 3G4 and AB815 antibodies (against the CAT and thehinge, respectively) were used inWestern blotting.MT1-MMPimmunoreactivity was not detected in the mock cell controltransfected with the original plasmid lacking the MT1-MMPinsert, whereas other cell types expressed the comparable levelof MT1-MMP. Naturally, the 3G4 antibody did not detect the�CAT construct and the degraded, �40-kDa, MT1-MMP spe-cies, which were lacking the CAT. In turn, the degraded specieswere absent in the inert E240A and �CAT mutants. Asexpected, the size of the degraded �PEX construct was �20kDa lower compared with the WT construct.The functional activity of cellular MT1-MMP was measured

using the ability of cells to activate the latentMMP-2 zymogen,a direct downstream target of MT1-MMP (4, 47, 55). BecauseMCF7 cells do not synthesize MMP-2 naturally, the purifiedMMP-2 proenzyme was added to the cells. As expected, mock,

E240A, and �CAT cells did not activate MMP-2, whereas allother cell types, including �PEX, PEX/MMP-2, and PEX/MMP-9 cells, readily activated MMP-2 and transformed thelatent 68-kDa zymogen into the 64-kDa intermediate and, pre-dominantly, the 62-kDa mature enzyme of MMP-2 (Fig. 2B).These results directly suggest that the original PEX of MT1-MMP is not crucial for the MMP-2-processing function ofMT1-MMP, and they agree well with the results of others (41,56–58).Mutations Do Not Affect the Internalization Rate of

MT1-MMP—Todeterminewhether themutations affected thecell surface presentation and internalization rate of MT1-MMP, we used cell immunostaining. To inactivate cellularMT1-MMP and block its self-degradation, prior to immuno-staining procedures the cells were cultured in the presence ofGM6001 (42). The cells were then fixed and permeabilized orleft untreated. The cells were next stained with the 3G4 anti-body that recognizes the CAT and, as a result, reacts with thefull-length proenzyme-enzyme species but not with thedegraded forms of MT1-MMP. Because the CAT was missingin the �CAT construct, �CAT cells were stained with theAB815 antibody against the hinge region of MT1-MMP.In agreement with the immunoblotting (Fig. 2B), there was

noMT1-MMP immunoreactivity in the mockMCF-7 cell con-trol. Immunostaining demonstrated the presence of the MT1-MMP immunoreactivity in all MT1-MMP constructs (Fig. 3A).

FIGURE 2. Expression of the MT1-MMP constructs. A, domain structure of MT1-MMP mutants. S, signal peptide; PRO, prodomain; CAT, catalytic domain;H, hinge region; PEX, hemopexin domain; ST, stalk region; TM, transmembrane domain; and CYTO, cytoplasmic tail. WT, wild-type full-length MT1-MMP; �CAT,�PEX, and �CYTO represent mutants lacking the sequence regions 112–284, 319 –508, and 563–582 (dotted lines), respectively; E240A, the catalytically inertmutant; PEX/MMP-2 and PEX/MMP-9 are the chimeras in which the original PEX was replaced by the PEX of MMP-2 and MMP-9, respectively. B, Western blottingand gelatin zymography of mock, WT, E240A, �CAT, �PEX, �CYTO, PEX/MMP-2, and PEX/MMP-9 cells. Purified pro-MMP-2 (0.15 nM) was added to the cells.Following a 24-h incubation in serum-free DMEM, gelatin beads were added to the medium aliquots to capture MMP-2. The captured samples were analyzedby gelatin zymography (bottom panel). The cells were lysed, and the lysate aliquots were analyzed by Western blotting with the MT1-MMP 3G4 and AB815antibodies, which recognize the CAT and the hinge region, respectively (two upper panels).

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Cell surface-associated MT1-MMP expression was especiallyevident in the non-permeabilized cells, whereas the predomi-nantly vesicularMT1-MMP immunoreactivity was observed inthe permeabilized cells. In agreement with our previous obser-vations (39) and in contrast to other constructs, the immuno-reactivity pattern of the permeabilized and non-permeabilized�CYTO cells was similar. Predominant association with thecaveolin-enriched lipid rafts and the resulting slow internaliza-tion rate via the caveolae pathway explain this immunostainingpattern of the �CYTO construct (41, 59). Based on theseresults, we conclude that all of the MT1-MMP constructs wedesigned are efficiently trafficked through the cell compart-ment and presented on the plasma membrane of MCF-7 cells.

To corroborate this conclusion, we compared the internal-ization rate of cellular MT1-MMP (Fig. 3B). The cells weresurface-biotinylated with membrane-impermeable, cleavable,EZ-Link NHS-SS-biotin. Biotinylation was followed by incuba-tion of the cells at 37 °C to initiate protein uptake. Cells werenext transferred on ice to arrest protein trafficking and thentreated with MESNA to release the biotin moiety from theresidual cell surface-associated MT1-MMP molecules. Thebiotin-labeled internalized MT1-MMP was protected fromMESNA. The biotin-labeled MT1-MMP pool was then cap-tured on streptavidin beads and then analyzed by Westernblotting using the 3G4 and AB815 antibodies. These tests dem-onstrated that a major portion of cell surface-associated MT1-

FIGURE 3. Cell surface presentation and internalization of the MT1-MMP constructs. A, immunostaining of mock, WT, E240A, �CAT, �PEX, �CYTO,PEX/MMP-2, and PEX/MMP-9 cells. The cells were grown in the presence of GM6001 on glass coverslips for 24 h. The cells were then fixed, permeabilized(�Triton X-100), or left untreated (�Triton X-100), and stained using the MT1-MMP 3G4 antibody raised against the CAT (red). The nuclei are DAPI-stained(blue). Bar, 10 �m. The star indicates the �CAT construct, which was stained using the MT1-MMP AB815 antibody raised against the hinge region. B, the uptakeof MT1-MMP by the cells. Cells were incubated for 24 h in DMEM-10% FBS with GM6001 and then surface-biotinylated with cleavable EZ-Link NHS-SS biotin. Thecells were next incubated for 25 min at 37 °C to allow cell surface-associated MT1-MMP to be internalized. Biotin-labeled MT1-MMP was captured on strepta-vidin beads and examined by Western blotting with the MT1-MMP 3G4 and AB815 antibodies (top and bottom panels, respectively). Where indicated, MESNAwas used to release a biotin moiety from the cell surface-associated proteins. Control, WT cells were treated as above but at 0 °C to demonstrate the quantitativeremoval of the biotin label by MESNA.

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MMP (except the �CYTO construct) was already internalizedfollowing a 25-min incubation. In contrast, only a small fractionof the �CYTO construct was protected from MESNA, thusconsistently suggesting that the �CYTOMT1-MMP was inef-ficiently internalized (39, 41, 59).Inhibition of MT1-MMP by TIMPs—We next evaluated the

inhibitory effect of TIMP-1 and TIMP-2 on the MT1-MMP-mediated activation of MMP-2. The similarly high inhibitoryactivity of the TIMP-1 and TIMP-2 samples we used was con-firmed using the purified individual CAT of MT6-MMP andthe Mca-PLGL-Dpa-AR-NH2 peptide as a substrate (Fig. 4A).According to our earlier data, TIMP-1 and TIMP-2 were simi-larly potent in the inhibition of the MT6-MMP proteolyticactivity (33). In agreement, a 5 molar excess of TIMP-1 orTIMP-2 over MT6-MMP was sufficient in our current tests toachieve a near complete inhibition of the proteolytic activity,thus suggesting the equal inhibitory potency of our inhibitorsamples. In sharp contrast, the purified individual CAT of

MT1-MMP retained its full proteolytic activity in the presenceof the high, 125 nM, TIMP-1 concentrations (at a 1:25 molarratio of MT1-MMP/TIMP-1), whereas no proteolytic activ-ity was recorded in the presence of a 1:5 molar ratioMT1-MMP/TIMP-2.In agreement with the MT1-MMP test system, 100 nM

TIMP-1 was incapable of affecting the ability of theWT,�PEX,and �CYTO MT1-MMP constructs to mediate activation ofMMP-2 (Fig. 4B). In turn, 100 nM TIMP-1 performed as apotent inhibitor of the chimeric PEX/MMP-2 and, especiallyPEX/MMP-9 constructs, in the MT1-MMP/MMP-2 cellularactivation system. As expected, TIMP-2 appended A-TIMP-2(because of its ability to replacewild-typeTIMP-2 in theTIMP-2�pro-MMP-2 complex) (60, 61), andGM6001 readily inhibitedall of the MT1-MMP constructs.These results are consistent with the effect of the inhibitors

on the self-proteolysis of cellularMT1-MMPand on the level ofthe degraded forms of the cellular proteinase (Fig. 4B). As

FIGURE 4. The role of the PEX. A, TIMP-1 and TIMP-2 are similarly potent in inhibiting MT6-MMP but not MT1-MMP. The individual CAT of MT1-MMP andMT6-MMP (5 nM each) was co-incubated with TIMP-1 and TIMP-2 at the indicated enzyme/inhibitor molar ratio. The residual activity of MT1-MMP (right panel)and of MT6-MMP (left panel) was measured using Mca-PLGL-Dpa-AR-NH2 (10 �M) as a substrate. The samples were measured in triplicate. The results werehighly reproducible without any significant day to day variations. Numbers above the bars indicate the residual activity in percent relative to a “no TIMP” control.A 1:25 molar ratio of MT1-MMP/TIMP-1 corresponds to the 125 nM TIMP-1 concentration in the samples. B, the effects of TIMP-1 and TIMP-2 on the activity ofMT1-MMP. Mock, WT, �PEX, �CYTO, PEX/MMP-2, and PEX/MMP-9 cells were incubated for 24 h in serum-free medium supplemented with the MMP-2proenzyme (0.5 nM). Where indicated, TIMP-1 (100 nM), TIMP-2 (100 nM), A-TIMP-2 (100 nM), and GM6001 (50 �M) were each added to the cells. Medium aliquotswere analyzed by gelatin zymography (left panels). Cells were lysed, and the lysates were analyzed using Western blotting with the MT1-MMP AB815 antibody(right panels).

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expected, GM6001 almost quantitatively repressedMT1-MMPself-proteolysis, and, as a result, only insignificant amounts ofthe degraded forms were generated. TIMP-2 (100 nM) alsorepressed, albeit less efficiently, the self-degradation of theWT,�CYTO, �PEX, PEX/MMP-2, and PEX/MMP-9 constructs,whereas 100 nMA-TIMP-2waswithout a significant effect. Theinhibitory effect of 100 nMTIMP-1was observed onlywith boththe PEX/MMP-2 and PEX/MMP-9 chimeric constructs. Over-all, the ability of TIMP-1 to inactivate the PEX/MMP-2 andPEX/MMP-9 chimeras is contrasting relative to the resistanceof the individual CAT of MT1-MMP to the similar concentra-tions of the inhibitor.Quantitative Assessment of the Inhibitory Potency of

TIMPs—To quantitatively assess the inhibition of MT1-MMP by TIMPs, we used MCF7-�3 cells transiently trans-fected with the WT and PEX/MMP-9 constructs. Cells wereco-incubated with both the MMP-2 proenzyme and theincreasing concentrations of TIMP-1 or TIMP-2. Followinggelatin zymography of the concentrated medium aliquots,the residual levels of the proenzyme and the generated levelsof the MMP-2 intermediate were measured using the digi-tized gel images (Fig. 5). In agreement with multiple earlierreports (reviewed in Refs. 8, 62, 63), the cellular WT con-struct was readily inhibited by TIMP-2. In contrast, the cel-lular WT activity was not significantly inhibited by TIMP-1.Thus, only a 20% inhibition of MT1-MMP was observed atTIMP-1 concentrations as high as 1000 nM.

In agreementwith our other results (Fig. 4), the PEX/MMP-9construct was significantly inhibited by both TIMP-1 and

TIMP-2 (Fig. 5). Thus, a near quantitative inhibition of PEX/MMP-9 was observed at the concentrations of TIMP-1 as lowas 100 nM. In turn, TIMP-2 was similarly effective against WTand PEX/MMP-9. Because TIMP-1 does not bind the PEXdomain of MMP-2, our inhibitory results, especially if com-bined together, cannot be explained only by the non-inhibitoryTIMP-1 binding with the PEX domain in the PEX/MMP-9 andPEX/MMP-2 chimeras.TIMP-1 Inhibits Gelatin Degradation by MT1-MMP

Chimeras—We next measured the ability of the cellularMT1-MMP constructs, including WT, PEX/MMP-2, andPEX/MMP-9 to degrade FITC-gelatin. Mock cells were usedas a control. Where indicated, TIMP-1, TIMP-2, or GM6001were added to the samples. In 16 h the cells were fixed,stained with the MT1-MMP antibody, and observed using afluorescence microscope. Gelatinolytic activity was detectedby the presence of the dark zones of digested gelatin on thefluorescent background of the intact FITC-gelatin (Fig. 6).WT, PEX/MMP-2, and PEX/MMP-9 cells readily de-

graded FITC-gelatin, whereas mock cells were clearly nega-tive. GM6001 and TIMP-2 each blocked gelatin degradationby WT, PEX/MMP-2, and PEX/MMP-9 cells. In turn,TIMP-1 inhibited only the PEX/MMP-2 and PEX/MMP-9chimeras and was without any effect on the WT cells. Thus,these results suggest that not only the ability of activatingMMP-2 and of self-proteolysis but also of the gelatinolyticactivity of the MT1-MMP chimeras is repressed by bothTIMPs.

FIGURE 5. Quantitative inhibition measurements of TIMP-1 and TIMP-2 against the MT1-MMP cellular constructs. Transiently transfected WT andPEX/MMP-9 MFC7-�3 cells (1 � 105; top and bottom panels, respectively) were incubated for 24 h in serum-free medium supplemented with the MMP-2proenzyme (0.15 nM), TIMP-1, or TIMP-2 (1–1000 nM each). The medium aliquots (0.1 ml each) were precipitated with acetone (1:4 v/v) for 18 h at �20 °C. Theprecipitates were collected by centrifugation and washed using ice-cold acetone. The samples were then dissolved in 2% SDS and analyzed by gelatinzymography. The gels were scanned, and the images were digitized. The levels of the MMP-2 proenzyme (68 kDa) and the MT1-MMP-generated intermediateform (64 kDa) of MMP-2 were measured in the gel images using ImageJ. The PEX/MMP-9 construct is less efficient in MMP-2 activation compared with WT. Asa result, the initial conversion of the MMP-2 proenzyme into the activation intermediate was close to 50% in the PEX/MMP-9 samples rather than 100% as in theWT samples.

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DISCUSSION

MT1-MMP, the first characterized, archetypemember of theMT-MMP family, was discovered as anMMP-2 cellular activa-tor (47, 64). Pro-tumorigenic MT1-MMP is a key proteinase incell migration, and its inhibitors are urgently required to com-bat multiple diseases, including malignancies. MT1-MMP isknown to be readily inhibited by TIMPs, excluding TIMP-1,whereas otherMMPs are similarly sensitive to the inhibition byTIMPs, including TIMP-1, -2, -3, and -4 (8, 14, 21). From prac-tical perspectives, this unique relation between TIMP-1 andMT1-MMP facilitates the discrimination of the latter fromother individual MMPs.Our structural studies provided a basis for the hypothesis

that the global fold and the relative positions of the PEX and theCAT manipulate the way TIMPs, including TIMP-1 andTIMP-2, interact with MMPs, including MT1-MMP. Overall,our computational analysis suggests that a noticeablemotion ofthe PEX relative to the CAT is required to allow an inhibitorycomplex with the TIMP moiety. In the absence of this motion,the loop 6 of the CT-TIMP clashes with the C-terminal regionof the first N-terminal propeller blade of the PEX, includingMMP-1 and MMP-2 (PDB 1FBL and 1CK7), and, most proba-bly, also MT1-MMP. As a result, the inhibitory NT-TIMP can-not interact in a productive way with the active site of the CAT

(Fig. 1 and supplemental Fig. S3). The interdomain dynamicsand the intrinsic protein flexibility of both MMPs and TIMPsseem to control their binding interface and play a role in themechanisms involved in MMP inhibition by TIMPs (14). Thissuggestion agrees well with the studies by others who suggestedthat conformational adaptations are required to avoid obstaclesfor interaction between the full-length TIMP-1 and the CAT ofMT1-MMP and MT3-MMP (65).These previously underexploited structural data allowed us

to hypothesize that the nature and the fold of the MMP’s PEXcontribute to the selectivity of MMP inhibition by TIMPs.These parameters are distinct and additional to the direct inter-action of the NT-TIMP with the MMP’s CAT. Conversely, wesuggested that, if the natural PEX is modified in MT1-MMP,the proteolytic activity of the resulting mutant may becomesensitive to TIMP-1. To test our hypothesis, we constructed the�PEX, �CAT, and �CYTO MT1-MMP truncations and thePEX-swapped constructs. In the latter, the PEX of MMP-2 andMMP-9 replaced the original PEX in theMT1-MMP structure.The wild-type and the catalytically inert E240A constructs ofMT1-MMP were used as controls.We then tested the functionality of the MT1-MMP con-

structs. These tests included the level of the MT1-MMPpresentation on the cell surface and the rate of internaliza-

FIGURE 6. In situ FITC-gelatin zymography. Mock, WT, PEX/MMP-2, and PEX/MMP-9 cells were seeded in serum-free DMEM on FITC-gelatin-coated coverslips.Where indicated, cells were incubated in the presence of TIMP-1 (100 nM), TIMP-2 (100 nM), and GM6001 (50 �M). In 16 h, cells on FITC-gelatin (green) were fixed,permeabilized, and stained with the MT1-MMP 3G4 antibody (red). The dark areas show the digested FITC-gelatin. The nuclei are stained with DAPI (blue). Bar,40 �m.

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tion inside the cells. In addition, we measured the ability ofthe constructs we designed to activate MMP-2, to self-de-grade, and to hydrolyze gelatin in situ and also their responseto TIMP-1, TIMP-2, and appended A-TIMP-2 with the inac-tivated inhibitory NT-TIMP (60). A broad-range hydroxa-mate inhibitor, GM6001, was used as a control in our inhib-itory tests.We determined that, in contrast to other MT1-MMP con-

structs, the PEX/MMP-2 and PEX/MMP-9 chimeras becamesensitive to the inhibition by both TIMP-1 and TIMP-2. Theseresults provide evidence that the presence of these PEX moi-eties in the MT1-MMP structure, but not the original PEX,allows both TIMP-1 and TIMP-2 to interact with the active sitein the CAT of MT1-MMP.It is possible that in the chimeras the unnatural PEX stabi-

lizes the interactions of the otherwise weak TIMP-1 inhibitorwith the CAT of MT1-MMP. The effects of the PEX are not asprominent for TIMP-2 because of its intrinsic high affinity tothe MT1-MMP’s CAT. Overall, it becomes increasingly clearthat there is an interplay between the CT-TIMP and the PEX inthe inhibitory mechanism of MMPs by TIMPs.The structural parameters that are involved in these dynamic

interactions are not precisely clear as yet. Nevertheless, ourbiochemical studies suggest that constraining the TIMP speci-ficity appears even more challenging than before and that thestructural parameters of the PEXofMMPs should be taken intoaccount for TIMP re-engineering to harness the therapeuticpotential of newMMP antagonists with constrained selectivity.The use of the model systems involving the inhibitoryNT-TIMP alone and the CAT of the individual MMPsmay notsatisfy the criteria that are required for the efficient and selec-tive inhibition of the full-length MMPs in vivo.

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Inhibition of MT1-MMP by TIMP-1 and TIMP-2

21012 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 23 • JUNE 10, 2011

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Boguslaw Stec and Alex Y. StronginAlbert G. Remacle, Sergey A. Shiryaev, Ilian A. Radichev, Dmitri V. Rozanov,

Metalloproteinases by Tissue Inhibitors of MetalloproteinasesDynamic Interdomain Interactions Contribute to the Inhibition of Matrix

doi: 10.1074/jbc.M110.200139 originally published online April 25, 20112011, 286:21002-21012.J. Biol. Chem. 

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