Abstract
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.
Highlights
Centrioles are ninefold symmetrical structures at the core of centrosomes and in the form of basal bodies, at the base of cilia, which when dysfunctional lead to a wide range of inherited diseases including ciliopathies and microcephaly, and which frequently show abnormalities in structure and number in cancer [1]
We recently found that the C-terminal part of Sas6 binds Gorab, a trans-Golgi-associated protein, whose human counterpart is mutated in the wrinkled skin disease, gerodermia osteodysplastica [27,28,29,30]
A crystal structure of Ana2’s C–C domain has been determined, the crystal structure of the full-length Ana2 has not been resolved. As both Ana2 and Gorab interact with the C-terminal part of Sas6, it becomes important to understand the relationship of these binding sites with each other
Summary
Centrioles are ninefold symmetrical structures at the core of centrosomes and in the form of basal bodies, at the base of cilia, which when dysfunctional lead to a wide range of inherited diseases including ciliopathies and microcephaly, and which frequently show abnormalities in structure and number in cancer [1]. Plk phosphorylates four conserved residues in the STAN motif in the C-terminal part of Ana leading to the recruitment of Sas by Ana2 [14,15,16,17]. A crystal structure of Ana2’s C–C domain has been determined, the crystal structure of the full-length Ana has not been resolved As both Ana and Gorab interact with the C-terminal part of Sas, it becomes important to understand the relationship of these binding sites with each other. We have used a combination of HDX-MS together with in vitro and in vivo binding assays and mutational studies to map the interacting surfaces within the Ana2–Sas complex This confirms the interaction of Ana2’s STAN motif with Sas and defines the region within Sas6’s C–C responsible for that interaction. It reveals that Sas can accommodate the binding of both Ana and Gorab through interactions at different sites
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