Characterization of the mutations disrupting CP110–CPAP interaction. (A) Schematic representation of the domain organization of full-length human CPAP and CP110. The minimal regions CPAP and CP110 that interact with each other are indicated. The domain nomenclature is as in Fig. 4, A and B. (B and C) Chemical crosslinking followed by mass spectrometry of CPAP-CC1/CP110-CC2. (B) Schematic representations of parallel (left) and antiparallel (right) arrangements of CPAP-CC1 and CP110-CC2 chains in the CPAP-CC1/CP110-CC2 heterodimer. Predicted heptad repeats or H are indicated in each chain. Observed inter-protein crosslinks between residues of CPAP-CC1 and CP110-CC2 are indicated by thin lines. (C) Normalized inter-protein crosslinks observed between CPAP-CC1 and CP110-CC2 in the CPAP-CC1/CP110-CC2 heterodimer. The heptad a and d position residues are shown in bold and are underlined. The CPAP-CC1 and CP110-CC2 residues that were mutated in this study are highlighted with asterisks. (D) SEC-MALS analysis of CPAP-CC1 L149A/K150A (magenta dashed lines), CP110-CC2 (green solid lines), and an equimolar mixture of CPAP-CC1 L149A/K150A and CP110-CC2 (black solid lines). (E) Co-immunoprecipitation of CEP97^CP110-GFP as bait and CPAP-N-mCh WT or mutant as prey from HEK293T cells using anti-GFP antibodies. (F and G) Analytical SEC analysis of CPAP-CC1 and CP110-CC2 variants. (F) Analytical SEC analysis of CP110-CC2 (green solid line) and CP110-CC2 R656A/L659A (dark green–dashed line). (G) Analytical SEC analysis of CPAP-CC1 (magenta line), CP110-CC2 R656A/L659A (dark green–dashed line), and an equimolar mixture of CPAP-CC1/CP110-CC2 R656A/L659A (black solid line). Source data are available for this figure: SourceData F5.