Tubulin-tyrosine ligase has a binding site on beta-tubulin: a two-domain structure of the enzyme.
Tubulin-tyrosine ligase has a binding site on beta-tubulin: a two-domain structure of the enzyme.
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DOI：
10.1083/jcb.104.4.1059
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发表时间：
1987-04
期刊：
影响因子：
--
通讯作者：
Weber K
中科院分区：
文献类型：
--
作者：
Wehland J;Weber K
微管蛋白-酪氨酸连接酶和α-β-微管蛋白形成紧密的复合体，可通过甘油梯度离心法进行监测。利用两种不同的连接酶单抗、几种亚基特异性微管蛋白单抗和化学交联，在β-微管蛋白上鉴定了一个连接酶结合部位。当用枯草杆菌菌素处理去除两个微管蛋白亚基的羧基末端结构域时，该位点被保留。当连接酶添加到携带单抗YL 1/2的α-β-微管蛋白中时，也形成连接酶-微管蛋白复合体，该抗体仅与酪氨酸化的α-微管蛋白的羧基末端结合。这里描述的β-微管蛋白结合部位解释了连接酶的极端底物专一性，它不作用于其他细胞蛋白或从去酪氨酸化的α-微管蛋白衍生的羧基末端多肽。该位点在微管和微管中的不同可及性似乎解释了为什么连接酶优先作用于未聚合的微管蛋白。连接酶暴露在V8-蛋白酶下，转化为镍的衍生物。这不具有酶活性，但仍与微管蛋白形成复合体。凝胶电泳法记录了30-kD和14-kD两个结构域，每个结构域在免疫和生化上都是不同的，似乎覆盖了整个分子。这两个结构域在生理条件下紧密地相互作用。30kD的结构域携带β-微管蛋白和三磷酸腺苷的结合位点。14-kD结构域可能形成催化部位的额外部分，因为它含有单抗ID3的表位，该表位抑制酶活性，但不抑制连接酶-微管蛋白复合体的形成。
Tubulin-tyrosine ligase and alpha beta-tubulin form a tight complex which is conveniently monitored by glycerol gradient centrifugation. Using two distinct ligase monoclonal antibodies, several subunit- specific tubulin monoclonal antibodies, and chemical cross-linking, a ligase-binding site was identified on beta-tubulin. This site is retained when the carboxy-terminal domains of both tubulin subunits are removed by subtilisin treatment. The ligase-tubulin complex is also formed when ligase is added to alpha beta-tubulin carrying the monoclonal antibody YL 1/2 which binds only to the carboxyl end of tyrosinated alpha-tubulin. The beta-tubulin-binding site described here explains the extreme substrate specificity of ligase, which does not act on other cellular proteins or carboxy-terminal peptides derived from detyrosinated alpha-tubulin. Differential accessibility of this site in tubulin and in microtubules seems to explain why ligase acts preferentially on unpolymerized tubulin. Ligase exposed to V8-protease is converted to a nicked derivative. This is devoid of enzymatic activity but still forms the complex with tubulin. Gel electrophoresis documents both 30- and a 14-kD domains, each which is immunologically and biochemically distinct and seems to cover the entire molecule. The two domains interact tightly under physiological conditions. The 30-kD domain carries the binding sites for beta-tubulin and ATP. The 14-kD domain can possibly form an additional part of the catalytic site as it harbors the epitope for the monoclonal antibody ID3 which inhibits enzymatic activity but not the formation of the ligase-tubulin complex.