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Thermodynamic Basis of Selectivity in the Interactions of Tissue Inhibitors of Metalloproteinases N-domains with Matrix Metalloproteinases-1, -3, and -14*

  • Haiyin Zou
    Affiliations
    Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida 33431
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  • Ying Wu
    Footnotes
    Affiliations
    Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida 33431
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  • Keith Brew
    Correspondence
    To whom correspondence should be addressed. Tel.: 561-297-0407; Fax: 561-297-2221.
    Affiliations
    Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida 33431
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant RO1 AR40994. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 Present address: Dept. of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260.
Open AccessPublished:March 31, 2016DOI:https://doi.org/10.1074/jbc.M116.720250
      The four tissue inhibitors of metalloproteinases (TIMPs) are potent inhibitors of the many matrixins (MMPs), except that TIMP1 weakly inhibits some MMPs, including MMP14. The broad-spectrum inhibition of MMPs by TIMPs and their N-domains (NTIMPs) is consistent with the previous isothermal titration calorimetric finding that their interactions are entropy-driven but differ in contributions from solvent and conformational entropy (ΔSsolv, ΔSconf), estimated using heat capacity changes (ΔCp). Selective engineered NTIMPs have potential applications for treating MMP-related diseases, including cancer and cardiomyopathy. Here we report isothermal titration calorimetric studies of the effects of selectivity-modifying mutations in NTIMP1 and NTIMP2 on the thermodynamics of their interactions with MMP1, MMP3, and MMP14. The weak inhibition of MMP14 by NTIMP1 reflects a large conformational entropy penalty for binding. The T98L mutation, peripheral to the NTIMP1 reactive site, enhances binding by increasing ΔSsolv but also reduces ΔSconf. However, the same mutation increases NTIMP1 binding to MMP3 in an interaction that has an unusual positive ΔCp. This indicates a decrease in solvent entropy compensated by increased conformational entropy, possibly reflecting interactions involving alternative conformers. The NTIMP2 mutant, S2D/S4A is a selective MMP1 inhibitor through electrostatic effects of a unique MMP-1 arginine. Asp-2 increases reactive site polarity, reducing ΔCp, but increases conformational entropy to maintain strong binding to MMP1. There is a strong negative correlation between ΔSsolv and ΔSconf for all characterized interactions, but the data for each MMP have characteristic ranges, reflecting intrinsic differences in the structures and dynamics of their free and inhibitor-bound forms.

      Introduction

      Isothermal titration calorimetry can dissect the sources of the free energy changes for protein interactions with other macromolecules and ligands (
      • Velázquez Campoy A.
      • Freire E.
      ITC in the post-genomic era….? Priceless.
      ). Measurements of the heat released or absorbed during the titration of a protein with a binding partner can quantify the enthalpy of binding (ΔH), association constant Ka, and stoichiometry (N) (
      • Velázquez Campoy A.
      • Freire E.
      ITC in the post-genomic era….? Priceless.
      ), allowing the calculation of the changes in free energy (ΔG) and entropy (ΔS) of binding. Titrations in buffers with different enthalpies of ionization quantify ionization changes linked to binding, whereas the heat capacity change for binding, ΔCp, measured as the temperature dependence of ΔH, can be used to estimate the change in solvation entropy on binding (ΔSsolv). These parameters can be correlated with the character of the interaction interface and differences in dynamics and solvation between the free and bound conformer populations (
      • Murphy K.P.
      • Freire E.
      Thermodynamics of structural stability and cooperative folding behavior in proteins.
      ,
      • Spolar R.S.
      • Livingstone J.R.
      • Record Jr., M.T.
      Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water.
      ,
      • Baker B.M.
      • Murphy K.P.
      Prediction of binding energetics from structure using empirical parameterization.
      ).
      Previously, we used isothermal titration calorimetry to elucidate the thermodynamic profiles of interactions between the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases-1 and -2 (NT1 and NT2)
      The abbreviations used are: NT1
      N-terminal domain of TIMP-1
      NT2
      N-terminal domain of TIMP-2
      MMP
      matrix metalloproteinase
      MMP1c
      collagenase 1 catalytic domain
      MMP3c
      stromelysin 1 catalytic domain
      MMP14c
      membrane-type metalloproteinase-1 catalytic domain
      TIMP
      tissue inhibitor of metalloproteinase
      N-TIMP
      N-terminal inhibitory domain of TIMP
      Kiapp
      apparent inhibition constant
      Bes
      2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid
      Aces
      2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid.
      and the catalytic domains of matrix metalloproteinases -1 and -3 (MMP1c and MMP3c) (
      • Arumugam S.
      • Gao G.
      • Patton B.L.
      • Semenchenko V.
      • Brew K.
      • Van Doren S.R.
      Increased backbone mobility in β-barrel enhances entropy gain driving binding of N-TIMP-1 to MMP-3.
      ,
      • Wu Y.
      • Wei S.
      • Van Doren S.R.
      • Brew K.
      Entropy increases from different sources support the high-affinity binding of the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases to the catalytic domains of matrix metalloproteinases-1 and -3.
      ). TIMP1 and TIMP2 are two of the four human TIMPs, endogenous, broad spectrum, slow binding, high affinity inhibitors of the 23 human MMPs and several disintegrin-metalloproteinases (
      • Brew K.
      • Nagase H.
      The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity.
      ). The MMPs catalyze the proteolysis of all polypeptide components of the extracellular matrix and are crucial for tissue remodeling, wound healing, embryo implantation, cell migration, shedding of cell surface proteins, and release of bioactive peptides (
      • Murphy G.
      • Nagase H.
      Progress in matrix metalloproteinase research.
      ,
      • Mott J.D.
      • Werb Z.
      Regulation of matrix biology by matrix metalloproteinases.
      ); the unregulated activities of various MMPs have been linked to many disease processes including arthritis, heart disease, and tumor metastasis (
      • Murphy G.
      • Nagase H.
      Progress in matrix metalloproteinase research.
      ).
      The two domains of vertebrate TIMPs are each cross-linked by three disulfide bonds. The larger N-terminal domain (NTIMP) carries the MMP inhibitory activity, and the C-terminal domain mediates interactions with other proteins, including some pro-MMPs (
      • Brew K.
      • Nagase H.
      The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity.
      ). Recombinant forms of NTIMPs are fully active as MMP inhibitors (
      • Huang W.
      • Suzuki K.
      • Nagase H.
      • Arumugam S.
      • Van Doren S.R.
      • Brew K.
      Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E. coli.
      ) and have been extensively used in crystallographic (
      • Iyer S.
      • Wei S.
      • Brew K.
      • Acharya K.R.
      Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1.
      ,
      • Wisniewska M.
      • Goettig P.
      • Maskos K.
      • Belouski E.
      • Winters D.
      • Hecht R.
      • Black R.
      • Bode W.
      Structural determinants of the ADAM inhibition by TIMP-3: crystal structure of the TACE-N-TIMP-3 complex.
      ), mutational (
      • Meng Q.
      • Malinovskii V.
      • Huang W.
      • Hu Y.
      • Chung L.
      • Nagase H.
      • Bode W.
      • Maskos K.
      • Brew K.
      Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1′ residue of substrate.
      ,
      • Hamze A.B.
      • Wei S.
      • Bahudhanapati H.
      • Kota S.
      • Acharya K.R.
      • Brew K.
      Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1: gelatinase-selective inhibitors.
      ,
      • Lee M.-H.
      • Rapti M.
      • Knaüper V.
      • Murphy G.
      Threonine 98, the pivotal residue of tissue inhibitor of metalloproteinases (TIMP)-1 in metalloproteinase recognition.
      ,
      • Wei S.
      • Chen Y.
      • Chung L.
      • Nagase H.
      • Brew K.
      Protein engineering of the tissue inhibitor of metalloproteinase 1 (TIMP-1) inhibitory domain. In search of selective matrix metalloproteinase inhibitors.
      ,
      • Wei S.
      • Kashiwagi M.
      • Kota S.
      • Xie Z.
      • Nagase H.
      • Brew K.
      Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-α-converting enzyme.
      ,
      • Bahudhanapati H.
      • Zhang Y.
      • Sidhu S.S.
      • Brew K.
      Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).
      ), and NMR studies (
      • Huang W.
      • Suzuki K.
      • Nagase H.
      • Arumugam S.
      • Van Doren S.R.
      • Brew K.
      Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E. coli.
      ,
      • Arumugam S.
      • Hemme C.L.
      • Yoshida N.
      • Suzuki K.
      • Nagase H.
      • Berjanskii M.
      • Wu B.
      • Van Doren S.R.
      TIMP-1 contact sites and perturbations of stromelysin 1 mapped by NMR and a paramagnetic surface probe.
      ,
      • Williamson R.A.
      • Carr M.D.
      • Frenkiel T. A
      • Feeney J.
      • Freedman R.B.
      Mapping the binding site for matrix metalloproteinase on the N-terminal domain of the tissue inhibitor of metalloproteinases-2 by NMR chemical shift perturbation.
      ,
      • Williamson R.A.
      • Muskett F.W.
      • Howard M.J.
      • Freedman R.B.
      • Carr M.D.
      The effect of matrix metalloproteinase complex formation on the conformational mobility of tissue inhibitor of metalloproteinases-2 (TIMP-2).
      ,
      • Wu B.
      • Arumugam S.
      • Gao G.
      • Lee G.I.
      • Semenchenko V.
      • Huang W.
      • Brew K.
      • Van Doren S.R.
      NMR structure of tissue inhibitor of metalloproteinases-1 implicates localized induced fit in recognition of matrix metalloproteinases.
      ) of TIMP·MMP interactions. The known structures of complexes between full-length TIMPs and MMP catalytic domains show that the main interaction interface of the TIMP is provided by the N-domain with a few peripheral contacts from the C-domain (
      • Iyer S.
      • Wei S.
      • Brew K.
      • Acharya K.R.
      Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1.
      ,
      • Wisniewska M.
      • Goettig P.
      • Maskos K.
      • Belouski E.
      • Winters D.
      • Hecht R.
      • Black R.
      • Bode W.
      Structural determinants of the ADAM inhibition by TIMP-3: crystal structure of the TACE-N-TIMP-3 complex.
      ,
      • Gomis-Ruth F.-X.
      • Maskos K.
      • Betz M.
      • Bergner A.
      • Huber R.
      • Suzuki K.
      • Yoshida N.
      • Nagase H.
      • Brew K.
      • Bourenkov G.B.
      • Bartunik H.
      • Bode W.
      Mechanism of the inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.
      ,
      • Fernandez-Catalan C.
      • Bode W.
      • Huber R.
      • Turk D.
      • Calvete J.J.
      • Lichte A.
      • Tschesche H.
      • Maskos K.
      Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.
      ,
      • Maskos K.
      • Lang R.
      • Tschesche H.
      • Bode W.
      Flexibility and variability of TIMP binding: x-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.
      ,
      • Grossman M.
      • Tworowski D.
      • Dym O.
      • Lee M.H.
      • Levy Y.
      • Murphy G.
      • Sagi I.
      The intrinsic protein flexibility of endogenous protein inhibitor TIMP-1 controls its binding interface and affects its function.
      ,
      • Batra J.
      • Robinson J.
      • Soares A.S.
      • Fields A.P.
      • Radisky D.C.
      • Radisky E.S.
      Matrix metalloproteinase-10 (MMP-10) interaction with tissue inhibitors of metalloproteinase TIMP-1 and TIMP-2; binding studies and crystal structure.
      ). N-TIMPs have an oligonucleotide/oligosaccharide-binding fold, a 5-stranded β-barrel structure with two small helices. The core of the MMP-binding site of TIMP is a surface ridge formed by the N-terminal five residues, 1C(T/S)C(V/A/S)P5 and the connector between β-strands C and D, residues that are linked by the Cys-1 to Cys-70 disulfide bond (
      • Brew K.
      • Nagase H.
      The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity.
      ,
      • Gomis-Ruth F.-X.
      • Maskos K.
      • Betz M.
      • Bergner A.
      • Huber R.
      • Suzuki K.
      • Yoshida N.
      • Nagase H.
      • Brew K.
      • Bourenkov G.B.
      • Bartunik H.
      • Bode W.
      Mechanism of the inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.
      ,
      • Fernandez-Catalan C.
      • Bode W.
      • Huber R.
      • Turk D.
      • Calvete J.J.
      • Lichte A.
      • Tschesche H.
      • Maskos K.
      Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.
      ,
      • Maskos K.
      • Lang R.
      • Tschesche H.
      • Bode W.
      Flexibility and variability of TIMP binding: x-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.
      ). As shown in Fig. 1, this region is oriented in the active site of the MMP so that the α-amino group and carbonyl oxygen of Cys-1 coordinate the catalytic zinc (
      • Iyer S.
      • Wei S.
      • Brew K.
      • Acharya K.R.
      Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1.
      ,
      • Wisniewska M.
      • Goettig P.
      • Maskos K.
      • Belouski E.
      • Winters D.
      • Hecht R.
      • Black R.
      • Bode W.
      Structural determinants of the ADAM inhibition by TIMP-3: crystal structure of the TACE-N-TIMP-3 complex.
      ,
      • Gomis-Ruth F.-X.
      • Maskos K.
      • Betz M.
      • Bergner A.
      • Huber R.
      • Suzuki K.
      • Yoshida N.
      • Nagase H.
      • Brew K.
      • Bourenkov G.B.
      • Bartunik H.
      • Bode W.
      Mechanism of the inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.
      ,
      • Fernandez-Catalan C.
      • Bode W.
      • Huber R.
      • Turk D.
      • Calvete J.J.
      • Lichte A.
      • Tschesche H.
      • Maskos K.
      Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.
      ,
      • Maskos K.
      • Lang R.
      • Tschesche H.
      • Bode W.
      Flexibility and variability of TIMP binding: x-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.
      ,
      • Grossman M.
      • Tworowski D.
      • Dym O.
      • Lee M.H.
      • Levy Y.
      • Murphy G.
      • Sagi I.
      The intrinsic protein flexibility of endogenous protein inhibitor TIMP-1 controls its binding interface and affects its function.
      ,
      • Batra J.
      • Robinson J.
      • Soares A.S.
      • Fields A.P.
      • Radisky D.C.
      • Radisky E.S.
      Matrix metalloproteinase-10 (MMP-10) interaction with tissue inhibitors of metalloproteinase TIMP-1 and TIMP-2; binding studies and crystal structure.
      ,
      • Batra J.
      • Soares A.S.
      • Mehner C.
      • Radisky E.S.
      Matrix metalloproteinase-10/TIMP-2 structure and analyses define conserved core interactions and diverse exosite interactions in MMP/TIMP complexes.
      ). When the α-amino group is chemically modified or extended by an N-terminal alanine, the MMP inhibitory activity is essentially eliminated (
      • Wei S.
      • Kashiwagi M.
      • Kota S.
      • Xie Z.
      • Nagase H.
      • Brew K.
      Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-α-converting enzyme.
      ,
      • Higashi S.
      • Miyazaki K.
      Reactive site-modified tissue inhibitor of metalloproteinases-2 inhibits the cell-mediated activation of progelatinase A.
      ,
      • Van Doren S.R.
      • Wei S.
      • Gao G.
      • DaGue B.B.
      • Palmier M.O.
      • Bahudhanapati H.
      • Brew K.
      Inactivation of N-TIMP-1 by N-terminal acetylation when expressed in bacteria.
      ). The side chain of TIMP residue 2 (Ser or Thr) sits over the mouth of the S1′ subsite (the “specificity pocket”) of the MMP active site (
      • Iyer S.
      • Wei S.
      • Brew K.
      • Acharya K.R.
      Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1.
      ,
      • Wisniewska M.
      • Goettig P.
      • Maskos K.
      • Belouski E.
      • Winters D.
      • Hecht R.
      • Black R.
      • Bode W.
      Structural determinants of the ADAM inhibition by TIMP-3: crystal structure of the TACE-N-TIMP-3 complex.
      ,
      • Gomis-Ruth F.-X.
      • Maskos K.
      • Betz M.
      • Bergner A.
      • Huber R.
      • Suzuki K.
      • Yoshida N.
      • Nagase H.
      • Brew K.
      • Bourenkov G.B.
      • Bartunik H.
      • Bode W.
      Mechanism of the inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.
      ,
      • Fernandez-Catalan C.
      • Bode W.
      • Huber R.
      • Turk D.
      • Calvete J.J.
      • Lichte A.
      • Tschesche H.
      • Maskos K.
      Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.
      ,
      • Maskos K.
      • Lang R.
      • Tschesche H.
      • Bode W.
      Flexibility and variability of TIMP binding: x-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.
      ,
      • Grossman M.
      • Tworowski D.
      • Dym O.
      • Lee M.H.
      • Levy Y.
      • Murphy G.
      • Sagi I.
      The intrinsic protein flexibility of endogenous protein inhibitor TIMP-1 controls its binding interface and affects its function.
      ,
      • Batra J.
      • Robinson J.
      • Soares A.S.
      • Fields A.P.
      • Radisky D.C.
      • Radisky E.S.
      Matrix metalloproteinase-10 (MMP-10) interaction with tissue inhibitors of metalloproteinase TIMP-1 and TIMP-2; binding studies and crystal structure.
      ,
      • Batra J.
      • Soares A.S.
      • Mehner C.
      • Radisky E.S.
      Matrix metalloproteinase-10/TIMP-2 structure and analyses define conserved core interactions and diverse exosite interactions in MMP/TIMP complexes.
      ), and eliminating the side chain by substitution with glycine greatly weakens the affinity for most, but not all, MMPs (
      • Meng Q.
      • Malinovskii V.
      • Huang W.
      • Hu Y.
      • Chung L.
      • Nagase H.
      • Bode W.
      • Maskos K.
      • Brew K.
      Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1′ residue of substrate.
      ,
      • Hamze A.B.
      • Wei S.
      • Bahudhanapati H.
      • Kota S.
      • Acharya K.R.
      • Brew K.
      Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1: gelatinase-selective inhibitors.
      ). Residues 3–5 interact with the S2′ and S3′ subsites, and the C-D β-strand connector interacts with the MMP S2 and S3 subsites (Fig. 1). Loops between β-strands A and B and strands E and F and the C-terminal end of β-strand D make variable contributions in different TIMP·MMP complexes (
      • Iyer S.
      • Wei S.
      • Brew K.
      • Acharya K.R.
      Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1.
      ,
      • Wisniewska M.
      • Goettig P.
      • Maskos K.
      • Belouski E.
      • Winters D.
      • Hecht R.
      • Black R.
      • Bode W.
      Structural determinants of the ADAM inhibition by TIMP-3: crystal structure of the TACE-N-TIMP-3 complex.
      ,
      • Gomis-Ruth F.-X.
      • Maskos K.
      • Betz M.
      • Bergner A.
      • Huber R.
      • Suzuki K.
      • Yoshida N.
      • Nagase H.
      • Brew K.
      • Bourenkov G.B.
      • Bartunik H.
      • Bode W.
      Mechanism of the inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.
      ,
      • Fernandez-Catalan C.
      • Bode W.
      • Huber R.
      • Turk D.
      • Calvete J.J.
      • Lichte A.
      • Tschesche H.
      • Maskos K.
      Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.
      ,
      • Maskos K.
      • Lang R.
      • Tschesche H.
      • Bode W.
      Flexibility and variability of TIMP binding: x-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.
      ,
      • Grossman M.
      • Tworowski D.
      • Dym O.
      • Lee M.H.
      • Levy Y.
      • Murphy G.
      • Sagi I.
      The intrinsic protein flexibility of endogenous protein inhibitor TIMP-1 controls its binding interface and affects its function.
      ,
      • Batra J.
      • Robinson J.
      • Soares A.S.
      • Fields A.P.
      • Radisky D.C.
      • Radisky E.S.
      Matrix metalloproteinase-10 (MMP-10) interaction with tissue inhibitors of metalloproteinase TIMP-1 and TIMP-2; binding studies and crystal structure.
      ,
      • Batra J.
      • Soares A.S.
      • Mehner C.
      • Radisky E.S.
      Matrix metalloproteinase-10/TIMP-2 structure and analyses define conserved core interactions and diverse exosite interactions in MMP/TIMP complexes.
      ). The A-B loop of NT2 includes seven more residues than that of NT1 and has multiple interactions with the MMP in its complexes with MMP14 (MT1·MMP), MMP13c, and MMP10c (
      • Fernandez-Catalan C.
      • Bode W.
      • Huber R.
      • Turk D.
      • Calvete J.J.
      • Lichte A.
      • Tschesche H.
      • Maskos K.
      Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.
      ,
      • Maskos K.
      • Lang R.
      • Tschesche H.
      • Bode W.
      Flexibility and variability of TIMP binding: x-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.
      ,
      • Batra J.
      • Robinson J.
      • Soares A.S.
      • Fields A.P.
      • Radisky D.C.
      • Radisky E.S.
      Matrix metalloproteinase-10 (MMP-10) interaction with tissue inhibitors of metalloproteinase TIMP-1 and TIMP-2; binding studies and crystal structure.
      ,
      • Batra J.
      • Soares A.S.
      • Mehner C.
      • Radisky E.S.
      Matrix metalloproteinase-10/TIMP-2 structure and analyses define conserved core interactions and diverse exosite interactions in MMP/TIMP complexes.
      ) that are absent from complexes of TIMP-1 with MMP1c, MMP3c, or MMP10c (
      • Iyer S.
      • Wei S.
      • Brew K.
      • Acharya K.R.
      Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1.
      ,
      • Gomis-Ruth F.-X.
      • Maskos K.
      • Betz M.
      • Bergner A.
      • Huber R.
      • Suzuki K.
      • Yoshida N.
      • Nagase H.
      • Brew K.
      • Bourenkov G.B.
      • Bartunik H.
      • Bode W.
      Mechanism of the inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.
      ,
      • Batra J.
      • Robinson J.
      • Soares A.S.
      • Fields A.P.
      • Radisky D.C.
      • Radisky E.S.
      Matrix metalloproteinase-10 (MMP-10) interaction with tissue inhibitors of metalloproteinase TIMP-1 and TIMP-2; binding studies and crystal structure.
      ).
      Figure thumbnail gr1
      FIGURE 1A schematic view of the interactions between the core of the NT1 interaction ridge and the active site and substrate binding subsites of MMP1. The N-terminal five residues of TIMP-1 are colored cyan, residues 67–70 and 98–99 are gray, the Cys-1–Cys-70 and Cys-3–Cys-99 disulfide bonds are yellow, oxygen atoms are red, and nitrogen atoms are blue. Residues 214–221 of MMP1 are represented by the blue ribbon, and the catalytic Zn2+ is represented by a purple sphere.
      TIMP-3 inhibits more metalloproteinases than the other TIMPs, including disintegrin-metalloproteinases such as ADAM-17 (a disintegrin and metalloproteinase-17), ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin domains), and ADAMTS-5 (
      • Brew K.
      • Nagase H.
      The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity.
      ), and these interactions are affected differently from those with MMPs by mutations in its reactive site (
      • Wei S.
      • Kashiwagi M.
      • Kota S.
      • Xie Z.
      • Nagase H.
      • Brew K.
      Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-α-converting enzyme.
      ,
      • Lim N.H.
      • Kashiwagi M.
      • Visse R.
      • Jones J.
      • Enghild J.J.
      • Brew K.
      • Nagase H.
      Reactive site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: biological and structural implications.
      ). The specificity of N-TIMPs for MMPs can also be modified by protein engineering to produce variants with enhanced or reduced selectivity for MMPs (
      • Meng Q.
      • Malinovskii V.
      • Huang W.
      • Hu Y.
      • Chung L.
      • Nagase H.
      • Bode W.
      • Maskos K.
      • Brew K.
      Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1′ residue of substrate.
      ,
      • Hamze A.B.
      • Wei S.
      • Bahudhanapati H.
      • Kota S.
      • Acharya K.R.
      • Brew K.
      Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1: gelatinase-selective inhibitors.
      ,
      • Lee M.-H.
      • Rapti M.
      • Knaüper V.
      • Murphy G.
      Threonine 98, the pivotal residue of tissue inhibitor of metalloproteinases (TIMP)-1 in metalloproteinase recognition.
      ,
      • Wei S.
      • Chen Y.
      • Chung L.
      • Nagase H.
      • Brew K.
      Protein engineering of the tissue inhibitor of metalloproteinase 1 (TIMP-1) inhibitory domain. In search of selective matrix metalloproteinase inhibitors.
      ,
      • Wei S.
      • Kashiwagi M.
      • Kota S.
      • Xie Z.
      • Nagase H.
      • Brew K.
      Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-α-converting enzyme.
      ,
      • Bahudhanapati H.
      • Zhang Y.
      • Sidhu S.S.
      • Brew K.
      Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).
      ) and have potential applications treating diseases linked to excess MMP activities. Previously, we identified NT2 variants that inhibit MMP1 preferentially over MMP3 using phage display in conjunction with positive and negative selection with MMP1c and MMP3c, respectively (
      • Bahudhanapati H.
      • Zhang Y.
      • Sidhu S.S.
      • Brew K.
      Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).
      ). MMP1 (fibroblast collagenase) and MMP3 (stromelysin 1) have been validated as a target and anti-target for cancer therapy mediated by MMP inhibition (
      • Overall C.M.
      • Kleifeld O.
      Towards third generation matrix metalloproteinase inhibitors for cancer therapy.
      ). The most selective mutant, S2D/S4A, has a nanomolar Ki for MMP-1 but does not inhibit MMP3c or MMP14c and weakly inhibits MMP2c, MMP7c, MMP8c, and MMP13c (
      • Bahudhanapati H.
      • Zhang Y.
      • Sidhu S.S.
      • Brew K.
      Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).
      ). Although other TIMPs are high affinity inhibitors of all MMPs, TIMP-1 is a poor inhibitor of the membrane-type MMPs (MMP14, MMP16, and MMP24) and MMP19. The T98L substitution in NT1 has been shown to enhance its affinity for MMP14 (2–3-fold), and additional substitutions, V4A and P6V, increase the affinity, reflected in a 10-fold improvement of the Ki in the triple mutant (
      • Lee M.-H.
      • Rapti M.
      • Knaüper V.
      • Murphy G.
      Threonine 98, the pivotal residue of tissue inhibitor of metalloproteinases (TIMP)-1 in metalloproteinase recognition.
      ,
      • Hamze A.B.
      • Wei S.
      • Bahudhanapati H.
      • Kota S.
      • Acharya K.R.
      • Brew K.
      Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1: gelatinase-selective inhibitors.
      ). A crystallographic structure of the complex of the triple mutant of NT1 with MMP14 has been determined (
      • Grossman M.
      • Tworowski D.
      • Dym O.
      • Lee M.H.
      • Levy Y.
      • Murphy G.
      • Sagi I.
      The intrinsic protein flexibility of endogenous protein inhibitor TIMP-1 controls its binding interface and affects its function.
      ).
      Here, we have investigated how the mutations in NT1 and NT2 that modify MMP selectivity affect the thermodynamics of N-TIMP·MMP interactions, focusing on the T98L mutation in NT1 with MMP3c and MMP14c and the S2D/S4A double substitution in NT2 (with MMP1c). The thermodynamic profiles of the interactions of these MMPs with WT NT1 and NT2 (
      • Wu Y.
      • Wei S.
      • Van Doren S.R.
      • Brew K.
      Entropy increases from different sources support the high-affinity binding of the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases to the catalytic domains of matrix metalloproteinases-1 and -3.
      ) show that all are driven by entropy increases. Entropy-driven binding has been observed in other proteins that bind to multiple targets, including thioredoxin and protein kinase A (
      • Palde P.B.
      • Carroll K.S.
      A universal entropy-driven mechanism for thioredoxin-target recognition.
      ,
      • Chang C.E.
      • McLaughlin W.A.
      • Baron R.
      • Wang W.
      • McCammon J.A.
      Entropic contributions and the influence of the hydrophobic environment in promiscuous protein-protein association.
      ). The solvent entropy change for the interaction, ΔSsolv, can be estimated from the heat capacity change, ΔCp (
      • Baldwin R.L.
      Temperature dependence of the hydrophobic interaction in protein folding.
      ). Using ΔSsolv and ΔSint, the conformation entropy change for the interaction (ΔSconf) can be determined to provide information about differences in conformational dynamics between the free and bound forms of the two proteins. The results highlight the contributions of changes in conformational dynamics and solvent entropy (the hydrophobic effect) to differences in binding to different MMPs.

      Author Contributions

      K. B. devised and supervised the study and wrote the paper. H. Z. and Y. W. designed and performed the experiments and analyzed the data.

      References

        • Velázquez Campoy A.
        • Freire E.
        ITC in the post-genomic era….? Priceless.
        Biophys. Chem. 2005; 115: 115-124
        • Murphy K.P.
        • Freire E.
        Thermodynamics of structural stability and cooperative folding behavior in proteins.
        Adv. Protein Chem. 1992; 43: 313-361
        • Spolar R.S.
        • Livingstone J.R.
        • Record Jr., M.T.
        Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water.
        Biochemistry. 1992; 31: 3947-3955
        • Baker B.M.
        • Murphy K.P.
        Prediction of binding energetics from structure using empirical parameterization.
        Methods Enzymol. 1998; 295: 294-315
        • Arumugam S.
        • Gao G.
        • Patton B.L.
        • Semenchenko V.
        • Brew K.
        • Van Doren S.R.
        Increased backbone mobility in β-barrel enhances entropy gain driving binding of N-TIMP-1 to MMP-3.
        J. Mol. Biol. 2003; 327: 719-734
        • Wu Y.
        • Wei S.
        • Van Doren S.R.
        • Brew K.
        Entropy increases from different sources support the high-affinity binding of the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases to the catalytic domains of matrix metalloproteinases-1 and -3.
        J. Biol. Chem. 2011; 286: 16891-16899
        • Brew K.
        • Nagase H.
        The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity.
        Biochim. Biophys. Acta. 2010; 1803: 55-71
        • Murphy G.
        • Nagase H.
        Progress in matrix metalloproteinase research.
        Mol. Aspects Med. 2008; 29: 290-308
        • Mott J.D.
        • Werb Z.
        Regulation of matrix biology by matrix metalloproteinases.
        Curr. Opin. Cell Biol. 2004; 16: 558-564
        • Huang W.
        • Suzuki K.
        • Nagase H.
        • Arumugam S.
        • Van Doren S.R.
        • Brew K.
        Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E. coli.
        FEBS Lett. 1996; 384: 155-161
        • Iyer S.
        • Wei S.
        • Brew K.
        • Acharya K.R.
        Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1.
        J. Biol. Chem. 2007; 282: 364-371
        • Wisniewska M.
        • Goettig P.
        • Maskos K.
        • Belouski E.
        • Winters D.
        • Hecht R.
        • Black R.
        • Bode W.
        Structural determinants of the ADAM inhibition by TIMP-3: crystal structure of the TACE-N-TIMP-3 complex.
        J. Mol. Biol. 2008; 381: 1307-1319
        • Meng Q.
        • Malinovskii V.
        • Huang W.
        • Hu Y.
        • Chung L.
        • Nagase H.
        • Bode W.
        • Maskos K.
        • Brew K.
        Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1′ residue of substrate.
        J. Biol. Chem. 1999; 274: 10184-10189
        • Hamze A.B.
        • Wei S.
        • Bahudhanapati H.
        • Kota S.
        • Acharya K.R.
        • Brew K.
        Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1: gelatinase-selective inhibitors.
        Protein Sci. 2007; 16: 1905-1913
        • Lee M.-H.
        • Rapti M.
        • Knaüper V.
        • Murphy G.
        Threonine 98, the pivotal residue of tissue inhibitor of metalloproteinases (TIMP)-1 in metalloproteinase recognition.
        J. Biol. Chem. 2004; 279: 17562-17569
        • Wei S.
        • Chen Y.
        • Chung L.
        • Nagase H.
        • Brew K.
        Protein engineering of the tissue inhibitor of metalloproteinase 1 (TIMP-1) inhibitory domain. In search of selective matrix metalloproteinase inhibitors.
        J. Biol. Chem. 2003; 278: 9831-9834
        • Wei S.
        • Kashiwagi M.
        • Kota S.
        • Xie Z.
        • Nagase H.
        • Brew K.
        Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-α-converting enzyme.
        J. Biol. Chem. 2005; 280: 32877-32882
        • Bahudhanapati H.
        • Zhang Y.
        • Sidhu S.S.
        • Brew K.
        Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).
        J. Biol. Chem. 2011; 286: 31761-31770
        • Arumugam S.
        • Hemme C.L.
        • Yoshida N.
        • Suzuki K.
        • Nagase H.
        • Berjanskii M.
        • Wu B.
        • Van Doren S.R.
        TIMP-1 contact sites and perturbations of stromelysin 1 mapped by NMR and a paramagnetic surface probe.
        Biochemistry. 1998; 37: 9650-9657
        • Williamson R.A.
        • Carr M.D.
        • Frenkiel T. A
        • Feeney J.
        • Freedman R.B.
        Mapping the binding site for matrix metalloproteinase on the N-terminal domain of the tissue inhibitor of metalloproteinases-2 by NMR chemical shift perturbation.
        Biochemistry. 1997; 36: 13882-13889
        • Williamson R.A.
        • Muskett F.W.
        • Howard M.J.
        • Freedman R.B.
        • Carr M.D.
        The effect of matrix metalloproteinase complex formation on the conformational mobility of tissue inhibitor of metalloproteinases-2 (TIMP-2).
        J. Biol. Chem. 1999; 274: 37226-37232
        • Wu B.
        • Arumugam S.
        • Gao G.
        • Lee G.I.
        • Semenchenko V.
        • Huang W.
        • Brew K.
        • Van Doren S.R.
        NMR structure of tissue inhibitor of metalloproteinases-1 implicates localized induced fit in recognition of matrix metalloproteinases.
        J. Mol. Biol. 2000; 295: 257-268
        • Gomis-Ruth F.-X.
        • Maskos K.
        • Betz M.
        • Bergner A.
        • Huber R.
        • Suzuki K.
        • Yoshida N.
        • Nagase H.
        • Brew K.
        • Bourenkov G.B.
        • Bartunik H.
        • Bode W.
        Mechanism of the inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.
        Nature. 1997; 389: 77-81
        • Fernandez-Catalan C.
        • Bode W.
        • Huber R.
        • Turk D.
        • Calvete J.J.
        • Lichte A.
        • Tschesche H.
        • Maskos K.
        Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.
        EMBO J. 1998; 17: 5238-5248
        • Maskos K.
        • Lang R.
        • Tschesche H.
        • Bode W.
        Flexibility and variability of TIMP binding: x-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.
        J. Mol. Biol. 2007; 366: 1222-1231
        • Grossman M.
        • Tworowski D.
        • Dym O.
        • Lee M.H.
        • Levy Y.
        • Murphy G.
        • Sagi I.
        The intrinsic protein flexibility of endogenous protein inhibitor TIMP-1 controls its binding interface and affects its function.
        Biochemistry. 2010; 49: 6184-6192
        • Batra J.
        • Robinson J.
        • Soares A.S.
        • Fields A.P.
        • Radisky D.C.
        • Radisky E.S.
        Matrix metalloproteinase-10 (MMP-10) interaction with tissue inhibitors of metalloproteinase TIMP-1 and TIMP-2; binding studies and crystal structure.
        J. Biol. Chem. 2012; 287: 15935-15946
        • Batra J.
        • Soares A.S.
        • Mehner C.
        • Radisky E.S.
        Matrix metalloproteinase-10/TIMP-2 structure and analyses define conserved core interactions and diverse exosite interactions in MMP/TIMP complexes.
        Plos ONE. 2013; 8: e75836
        • Higashi S.
        • Miyazaki K.
        Reactive site-modified tissue inhibitor of metalloproteinases-2 inhibits the cell-mediated activation of progelatinase A.
        J. Biol. Chem. 1999; 274: 10497-10504
        • Van Doren S.R.
        • Wei S.
        • Gao G.
        • DaGue B.B.
        • Palmier M.O.
        • Bahudhanapati H.
        • Brew K.
        Inactivation of N-TIMP-1 by N-terminal acetylation when expressed in bacteria.
        Biopolymers. 2008; 89: 960-968
        • Lim N.H.
        • Kashiwagi M.
        • Visse R.
        • Jones J.
        • Enghild J.J.
        • Brew K.
        • Nagase H.
        Reactive site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: biological and structural implications.
        Biochem. J. 2010; 431: 113-122
        • Overall C.M.
        • Kleifeld O.
        Towards third generation matrix metalloproteinase inhibitors for cancer therapy.
        Br. J. Cancer. 2006; 94: 941-946
        • Palde P.B.
        • Carroll K.S.
        A universal entropy-driven mechanism for thioredoxin-target recognition.
        Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 7960-7965
        • Chang C.E.
        • McLaughlin W.A.
        • Baron R.
        • Wang W.
        • McCammon J.A.
        Entropic contributions and the influence of the hydrophobic environment in promiscuous protein-protein association.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 7456-7461
        • Baldwin R.L.
        Temperature dependence of the hydrophobic interaction in protein folding.
        Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 8069-8072
        • Spolar R.S.
        • Record Jr, M.T.
        Coupling of local folding to site-specific binding of proteins to DNA.
        Science. 1994; 263: 777-784
        • Negi S.S.
        • Schein C.H.
        • Oezguen N.
        • Power T.D.
        • Braun W.
        InterProSurf: a web server for predicting interacting sites on protein surfaces.
        Bioinformatics. 2007; 23: 3397-3399
        • Kumar S.
        • Ma B.
        • Tsai C.J.
        • Sinha N.
        • Nussinov R.
        Folding and binding cascades: dynamic landscapes and population shifts.
        Protein Sci. 2000; 9: 10-19
        • Grünberg R.
        • Nilges M.
        • Leckner J.
        Flexibility and conformational entropy in protein-protein binding.
        Structure. 2006; 14: 683-693
        • Eftink M.R.
        • Anusiem A.C.
        • Biltonen R.L.
        Enthalpy-entropy compensation and heat capacity changes for protein-ligand interactions: general thermodynamic models and data for the binding of nucleotides to ribonuclease A.
        Biochemistry. 1983; 22: 3884-3896
        • Liang X.
        • Arunima A.
        • Zhao Y.
        • Bhaskaran R.
        • Shende A.
        • Byrne T.S.
        • Fleeks J.
        • Palmier M.O.
        • Van Doren S.R.
        Apparent tradeoff of higher activity in MMP-12 for enhanced stability and flexibility in MMP-3.
        Biophys. J. 2010; 99: 273-283
        • Chen L.
        • Rydel T.J.
        • Gu F.
        • Dunaway C.M.
        • Pikul S.
        • Dunham K.M.
        • Barnett B.L.
        Crystal structure of the stromelysin catalytic domain at 2.0 Å resolution: inhibitor-induced conformational changes.
        J. Mol. Biol. 1999; 293: 545-557
        • Pettersen E.F.
        • Goddard T.D.
        • Huang C.C.
        • Couch G.S.
        • Greenblatt D.M.
        • Meng E.C.
        • Ferrin T.E.
        UCSF Chimera-a visualization system for exploratory research and analysis.
        J. Comput. Chem. 2004; 25: 1605-1612
        • Prabhu N.V.
        • Sharp K.A.
        Heat capacity in proteins.
        Annu Rev. Phys. Chem. 2005; 56: 521-548
        • Erijman A.
        • Aizner Y.
        • Shifman J.M.
        Multispecific recognition: mechanism, evolution, and design.
        Biochemistry. 2011; 50: 602-611