Phosphorylation of Ana2 promotes stronger binding to Sas4 but not in the phospho-null S38A mutant. (A) Calculated dissociation constants measured by bio-layer interferometry. Soluble WT Ana2 (FL or amino acids 1–60) or mutant S38A were either untreated or prephosphorylated by Plk4 and then incubated with probe-attached Sas4 G-box (amino acids 606–901; see Fig. S3 and Materials and methods). (B) I-TASSER protein structure predictions of WT (top) and S38D (bottom) Ana2. C-scores are shown. Note that the single S38D PM substitution is predicted to dramatically alter Ana2 conformation, folding Ana2 so that the N and C termini are closely positioned. (C) I-TASSER predictions of secondary structure within Ana2. The position of WT S38 (upper panel; dotted line) and PM S38D (lower panel) is indicated in a short segment that changes from an α-helix to a β-strand. (D) The PM (5PM) FL Ana2 construct interacts strongly with FL Sas4 by Y2H analysis. WT and 5PM Ana2 were screened against Sas4. In each image, colonies from replica plating are shown, and growth indicates the presence of bound bait and prey. From left to right: (1) no selection; (2) growth selection on QDO; growth and color selection on (3) DDOXA and (4) QDOXA (blue color indicates an interaction). See also section Y2H assay in Materials and methods.