Francis Joshua W, Goswami Devrishi, Novick Scott J, Pascal Bruce D, Weikum Emily R, Ortlund Eric A, Griffin Patrick R, Kahn Richard A
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, United States.
Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33458, United States.
J Mol Biol. 2017 Nov 24;429(23):3696-3716. doi: 10.1016/j.jmb.2017.09.016. Epub 2017 Sep 29.
Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αβ-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A-E (TBCA-E), and GTP are required for the folding of α- and β-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCD•ARL2•β-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each's role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not β-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide-binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCD•ARL2•β-tubulin complex represents a functional intermediate in the β-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the β-tubulin in the trimer is also shown, with implications to modeling the pathway.
微管是高度动态的微管蛋白聚合物，参与多种细胞功能。尽管细胞中微管蛋白二聚体群体很重要，但我们对αβ-微管蛋白异二聚体生物合成所涉及的机制了解并不完整。除了前折叠蛋白和TCP-1环复合物外，α-和β-微管蛋白亚基折叠并组装成异二聚体还需要五种微管蛋白特异性伴侣蛋白，称为辅助因子A-E（TBCA-E）和GTP。我们最近描述了一种新型三聚体TBCD•ARL2•β-微管蛋白的纯化。在这里，我们采用氢/氘交换结合质谱法来探索三聚体中每种蛋白质的动力学。添加鸟嘌呤核苷酸导致每种蛋白质区域的溶剂可及性发生变化，从而对其在微管蛋白折叠中的作用做出预测。对该模型的初步测试证实，在三聚体中交换GTP的是ARL2，而不是β-微管蛋白。将ARL2单体与三聚体中的ARL2的动力学进行比较表明，其蛋白质相互作用与具有效应器的典型GTP酶相当。核苷酸结合测定结果支持了这一点，该测定揭示了三聚体中ARL2对GTP的亲和力增加。我们得出结论，TBCD•ARL2•β-微管蛋白复合物代表β-微管蛋白折叠途径中的一种功能中间体，其活性受ARL2上核苷酸循环的调节。还展示了三聚体中β-微管蛋白上鸟嘌呤核苷酸的共纯化，这对该途径的建模具有启示意义。
