Control of the structural stability of the tubulin dimer by one high affinity bound magnesium ion at nucleotide N-site
Control of the structural stability of the tubulin dimer by one high affinity bound magnesium ion at nucleotide N-site
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DOI：
10.1074/jbc.273.1.167
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发表时间：
1998-01-02
影响因子：
4.8
通讯作者：
Andreu, JM
中科院分区：
文献类型：
--
作者：
Menéndez, M;Rivas, G;Andreu, JM
α亚基N位GTP和β亚基E位GTP配基的微管蛋白(GDP-tuBulin)可逆地与一个高亲和力的镁离子结合(K-b=1.1×10(7)M-1)，而两个亚基的GTP配基的微管蛋白(GTP-tuBulin)又与另一个高亲和力的镁离子结合。这两个阳离子结合位点分别被鉴定为核苷酸位点N和E。N位的镁离子控制着α-β-微管蛋白二聚体的稳定性和结构。镁离子解离之后是结合核苷酸的缓慢释放和功能失活。结合到N位的镁离子显著增加了GDP-微管蛋白二聚体的热稳定性(提高了10摄氏度，类似于50千卡摩尔(-1)的实验热变)。然而，以镁离子为配体的GDP-微管蛋白和GTP-微管蛋白的热稳定性是相同的。镁离子与N-位的结合与α-β-二聚体的形成有关。结合镁离子，α-亚基向与β-亚基结合的秋水仙素类似物传递显著的荧光增强。秋水仙碱反过来又能稳定耗尽镁离子的微管蛋白。如果N-位和秋水仙碱位在α-β二聚界面，所描述的微管蛋白性质将被简单地解释。因此，E-位点将位于微管蛋白二聚体的β-端，这与E核苷酸γ-磷酸和配位阳离子控制微管稳定性的已知功能一致。
Tubulin liganded with GTP at the N-site in the alpha-subunit and with GDP at the E-site in the beta-subunit (GDP-tubulin) reversibly binds one high affinity Mg2+ cation (K-b = 1.1 x 10(7) M-1), whereas tubulin liganded with GTP at both subunits (GTP-tubulin) binds one more high affinity Mg2+. The two cation binding loci are identified as nucleotide sites N and E, respectively. Mg2+ at the N-site controls the stability and structure of the alpha beta-tubulin dimer. Mg2+ dissociation is followed by the slow release of bound nucleotide and functional inactivation. Mg2+ bound to the N-site significantly increases the thermal stability of the GDP-tubulin dimer (by 10 degrees C and similar to 50 kcal mol(-1) of experimental enthalpy change). However, the thermal stability of Mg2+-liganded GDP- and GTP-tubulin is the same. Mg2+ binding to the N-site is linked to the alpha beta-dimer formation. The binding of Mg2+, the alpha-subunit communicates a marked enhancement of fluorescence to a colchicine analogue bound to the beta-subunit. Colchicine, in turn, thermally stabilizes Mg2+-depleted tubulin. The tubulin properties described would be simply explained if the N-site and the colchicine site are at the alpha-beta dimerization interface. It follows that the E-site would be at the beta-end of the tubulin dimer, consistent with the known functional role of the E nucleotide gamma-phosphate and coordinated cation controlling microtubule stability.