Molecular interactions between matrilysin and the matrix metalloproteinase inhibitor doxycycline investigated by deuterium exchange mass spectrometry
Molecular interactions between matrilysin and the matrix metalloproteinase inhibitor doxycycline investigated by deuterium exchange mass spectrometry
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DOI：
10.1124/mol.104.006346
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发表时间：
2005-04-01
影响因子：
3.6
通讯作者：
Villarreal, FJ
中科院分区：
文献类型：
--
作者：
García, RA;Pantazatos, DP;Villarreal, FJ
基质金属蛋白酶（MMPs）在正常和病理性细胞外基质降解中起重要作用。氘交换质谱法（DXMS）用于定位广谱MMP抑制剂多西环素的活性形式的基质溶素（残基95-267）的结合区域，并评估多西环素结合诱导的结构变化。基质溶解素的结合底物与无抑制剂形式的DXMS分析揭示了位于结构锌结合位点侧翼的两个主要的氢/氘交换减少位点（残基145-153;残基193-204）。多西环素matrilysin结合的平衡透析研究产生了一个K-D为73 μ M的结合化学计量的2.3抑制剂分子每蛋白质，比较以及与DXMS的结果，显示主要减少氘交换在两个网站。较小的变化，氘交换明显的氨基和羧基末端是由于信使诱导的结构波动。色氨酸荧光猝灭实验matrilysin与碘化钾表明，多西环素结合诱导的构象变化。在多西环素的存在下，色氨酸淬灭相对于无底物基质溶解素减少约17%。基质溶素的X射线晶体结构的检查表明，在残基193至204处的强力霉素结合位点位于基质溶素的结构金属中心内，邻近结构锌原子和两个钙原子。这些结果表明多西环素抑制基质溶解素的模式，可能涉及与结构锌原子和/或钙原子的蛋白质的结构金属中心内的相互作用。
Matrix metalloproteinases (MMPs) play an essential role in normal and pathological extracellular matrix degradation. Deuterium exchange mass spectrometry (DXMS) was used to localize the binding regions of the broad-spectrum MMP inhibitor doxycycline on the active form of matrilysin ( residues 95-267) and to assess alterations in structure induced by doxycycline binding. DXMS analyses of inhibitor-bound versus inhibitor- free forms of matrilysin reveal two primary sites of reduced hydrogen/deuterium exchange ( residues 145-153; residues 193-204) that flank the structural zinc binding site. Equilibrium dialysis studies of doxycycline-matrilysin binding yielded a K-d of 73 mu M with a binding stoichiometry of 2.3 inhibitor molecules per protein, which compares well with DXMS results that show principal reduction in deuterium exchange at two sites. Lesser changes in deuterium exchange evident at the amino and carboxyl termini are attributed to inhibitor-induced structural fluctuations. Tryptophan fluorescence quenching experiments of matrilysin with potassium iodide suggest changes in conformation induced by doxycycline binding. In the presence of doxycycline, tryptophan quenching is reduced by approximately 17% relative to inhibitor-free matrilysin. Examination of the X-ray crystal structure of matrilysin shows that the doxycycline-binding site at residues 193 to 204 is positioned within the structural metal center of matrilysin, adjacent to the structural zinc atom and near both calcium atoms. These results suggest a mode of matrilysin inhibition by doxycycline that could involve interactions with the structural zinc atom and/or calcium atoms within the structural metal center of the protein.