Kai Heidi S-T, Butler Georgina S, Morrison Charlotte J, King Angela E, Pelman Gayle R, Overall Christopher M
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.
J Biol Chem. 2002 Dec 13;277(50):48696-707. doi: 10.1074/jbc.M209177200. Epub 2002 Oct 8.
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.
金属蛋白酶组织抑制剂（TIMP）-4与前基质金属蛋白酶（MMP）-2结合，并有效抑制MT1-MMP，但与TIMP-2不同的是，它既不形成三分子复合物，也不支持前MMP-2的激活。为了研究这两种TIMP之间的结构和功能差异，将C末端结构域（C-TIMP-4和C-TIMP-2）与其N结构域独立表达，并将突变引入C末端尾巴。肌红蛋白被用作一种新型重组融合蛋白伴侣，因为即使在复杂的蛋白质混合物中，通过光谱测量408nm处血红素Soret吸光度也能轻松计算出红色重组蛋白的摩尔当量。C-TIMP-4和C-TIMP-2都能结合前MMP-2并阻断伴刀豆球蛋白A诱导的该酶的细胞激活。对结合速率常数（k(on)）的测量表明，TIMP-4对MMP-2的抑制作用之前存在C结构域对接相互作用，但与TIMP-2不同的是，这种相互作用不会因C末端尾巴相互作用而增强，因此发生速率较慢。实际上，C-TIMP-4的结合稳定性不会因C末端尾巴的缺失而改变，但用TIMP-2的尾巴替换会使其对前MMP-2的亲和力增加约2倍，用TIMP-2 C末端尾巴酸性残基替换C-TIMP-4尾巴中的残基（V193E/Q194D）也会如此。相反，用TIMP-4的尾巴替换C-TIMP-2的C末端尾巴会使前MMP-2的结合减少约75%，将其酸性特征突变为相应的TIMP-4氨基酸残基（E192V/D193Q）也会使其减少。总之，这表明Glu(192)和Asp(193)在TIMP-2与前MMP-2结合中的重要性；TIMP-4 C末端尾巴中缺乏这些酸性残基，这降低了与MMP-2血红素结合蛋白C结构域形成复合物的稳定性，可能使TIMP-4无法支持前MMP-2的激活。
