The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled–coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 in vitro and in vivo. Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2–Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously.

中心粒是一个九重对称结构，位于中心体的核心，作为基体，位于纤毛的基部，其保守复制受 Plk4 激酶调节。Plk4 磷酸化 Ana2 N 末端的单个丝氨酸残基，以促进 Ana2 加载到原中心粒形成位点。Ana2 的 STAN 基序中的四个保守丝氨酸随后被 Plk4 磷酸化，从而启用 Sas6 募集。晶体学数据表明 Ana2 的卷曲螺旋结构域形成四聚体，但全长 Ana2 的结构尚未解析。在这里，我们采用氢-氘交换结合质谱 (HDX-MS) 来揭示 Ana2 的构象动力学，揭示了这种蛋白质具有一个刚性区域的高度灵活性。体外和体内。通过 HDX-MS 对该复合物的分析确定了这种相互作用所需的短关键区域，这些区域位于两种蛋白质的 C 末端部分。突变研究证实了这些区域与 Ana2–Sas6 相互作用的相关性。Ana2 结合所需的 Sas6 位点不同于 Sas6 结合 Gorab 所需的位点，并且 Sas6 能够同时结合这两种蛋白质伙伴。