Identification of the extracellular matrix (ECM) binding motifs of tissue inhibitor of metalloproteinases (TIMP)-3 and effective transfer to TIMP-1.
Tissue inhibitor of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent endopeptidases of the matrix metalloproteinase families. There are four mammalian TIMPs (TIMP-1 to -4) but only TIMP-3 is sequestered to the extracellular matrix (ECM). The molecular basis for the TIMP-3:ECM association has never been fully investigated until now. In this report, we identify the unique amino acid configuration that constitutes the basis of the ECM binding motif in TIMP-3. By systematically exchanging the subdomains of the TIMPs and exhaustive mutation of TIMP-3, we have identified the surface residues directly responsible for ECM association. Contrary to the accepted view, we have found that TIMP-3 interacts with the ECM via both its N- and C-terminal domains. The amino acids involved in ECM binding are all basic in nature: Lys-26, Lys-27, Lys-30, Lys-76 of the N-terminal domain and Arg-163, Lys-165 of the C-terminal domain. Replacement of these residues with glutamate (E) and glutamine (Q) (K26/27/30/76E + R163/K165Q) resulted in a soluble TIMP-3 devoid of ECM-adhering ability. Using the ECM binding motif derived from TIMP-3, we have also created a TIMP-1 mutant (K26/27/30 + K76 transplant) capable of ECM association. This is the first instance of TIMPs being intentionally rendered soluble or ECM-bound. The ability to prepare TIMPs in soluble or ECM-bound forms also opens new avenues for future TIMP research.
Lee
Meng-Huee
MH
Department of Oncology, Cancer Research UK Cambridge Research Institute, Cambridge University, Cambridge CB2 0RE, United Kingdom.
Atkinson
Susan
S
Murphy
Gillian
G
D020816:Amino Acid Motifs; D001665:Binding Sites; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D008239:Lysine; D016296:Mutagenesis; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D012995:Solubility; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry / Q000378:metabolism*; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000737:chemistry / Q000378:metabolism*
D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D008239:Lysine