title: Utilization of a novel recombinant myoglobin fusion protein expression system to characterize the tissue inhibitor of metalloproteinase (TIMP)-4 and TIMP-2 C-terminal domain and tails by mutagenesis. The importance of acidic residues in binding the MMP-2 hemopexin C-domain.
abstract: Tissue inhibitor of metalloproteinase (TIMP)-4 binds pro-matrix metalloproteinase (MMP)-2 and efficiently inhibits MT1-MMP, but unlike TIMP-2 neither forms a trimolecular complex nor supports pro-MMP-2 activation. To investigate the structural and functional differences between these two TIMPs, the C-terminal domains (C-TIMP-4 and C-TIMP-2) were expressed independently from their N domains and mutations were introduced into the C-terminal tails. Myoglobin was used as a novel recombinant fusion protein partner because spectroscopic measurement of the heme Soret absorbance at 408 nm readily enabled calculation of the molar equivalent of the red-colored recombinant protein, even in complex protein mixtures. Both C-TIMP-4 and C-TIMP-2 bound pro-MMP-2 and blocked concanavalin A-induced cellular activation of the enzyme. Measurement of k(on) rates revealed that the inhibition of MMP-2 by TIMP-4 is preceded by a C domain docking interaction, but in contrast to TIMP-2, this is not enhanced by a C-terminal tail interaction and so occurs at a slower rate. Indeed, the binding stability of C-TIMP-4 was unaltered by deletion of the C-terminal tail, but replacement with the tail of TIMP-2 increased its affinity for pro-MMP-2 by approximately 2-fold, as did substitution with the TIMP-2 C-terminal tail acidic residues in the tail of C-TIMP-4 (V193E/Q194D). Conversely, substitution of the C-terminal tail of C-TIMP-2 with that of TIMP-4 reduced pro-MMP-2 binding by approximately 75%, as did reduction of its acidic character by mutation to the corresponding TIMP-4 amino acid residues (E192V/D193Q). Together, this shows the importance of Glu(192) and Asp(193) in TIMP-2 binding to pro-MMP-2; the lack of these acidic residues in the TIMP-4 C-terminal tail, which reduces the stability of complex formation with the MMP-2 hemopexin C domain, probably precludes TIMP-4 from supporting the activation of pro-MMP-2.
authors.affiliation: Canadian Institute of Health Research Group in Matrix Dynamics and the Department of Oral Biological and Medical Sciences, Faculty of Dentistry, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Heidi S-T
authors.initials: HS
authors.lastname: Kai
authors.forename: Georgina S
authors.initials: GS
authors.lastname: Butler
authors.forename: Charlotte J
authors.initials: CJ
authors.lastname: Morrison
authors.forename: Angela E
authors.initials: AE
authors.lastname: King
authors.forename: Gayle R
authors.initials: GR
authors.lastname: Pelman
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D006466:Hemopexin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019714:Tissue Inhibitor of Metalloproteinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097792:Tissue Inhibitor of Metalloproteinase-4
chemical_list: D017931:DNA Primers; D006466:Hemopexin; D020778:Matrix Metalloproteinase 2; D009211:Myoglobin; D011993:Recombinant Fusion Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D019714:Tissue Inhibitor of Metalloproteinases; D000097792:Tissue Inhibitor of Metalloproteinase-4