Figure 4.
Characterization of the CPAP–CP110 interaction. (A and B) Schemes of CP110 and CPAP illustrating the deletion mutants used in this study. “+,” interaction between CPAP and CP110; “−,” no interaction between CPAP and CP110, and “+/−,” weak interaction between CPAP and CP110. For CP110, CC1, and CC2 are the coiled-coil domains. For CPAP, CC1, and CC2 are coiled-coil domains; PN2–3, the tubulin-binding domain (Cormier et al., 2009); MBD, the MT-binding domain; and G-box, glycine-rich C-terminal domain forming an antiparallel β-sheet (Hatzopoulos et al., 2013). (C and D) Streptavidin pull-down assays with BioGFP-CP110 truncations as bait and full-length GFP-CPAP as prey. (E and F) Streptavidin pull-down assays with BioGFP-CPAP truncations as bait and full-length GFP-CP110 (E) or GFP-CP110 (581–991) (F) as prey. The assays in C–F were performed with extracts of HEK293T cells co-expressing the indicated constructs and BirA and analyzed by western blotting with anti-GFP antibodies. (G) SEC-MALS analysis of CPAP-CC1 (magenta line), CP110-CC2 (green line), and an equimolar mixture of CPAP-CC1 and CP110-CC2 (black line). (H) Scheme illustrating the mechanism for CPAP-CC1 and CP110-CC2 association. (I and J) CD spectra (I) recorded at 15°C and thermal-unfolding profiles (J) recorded by CD at 222 nm. Proteins and colors as in G. (K and L) SAXS analysis of the CPAP-CC1/CP110-CC2 heterodimer. (K) Solution X-ray scattering intensity over scattering angle from a 1:1 mixture (monomer equivalents) of CPAP-CC1 and CP110-CC2. The fit to the data yielding the interatomic distance distribution is shown with a black line. (L) Surface representation of the X-ray scattering volume of CPAP-CC1/CP110-CC2, at 32 ± 3 Å estimated precision, derived from averaging 22 particle models calculated by ab initio fit to the scattering data. Source data are available for this figure: SourceData F4.

Characterization of the CPAP–CP110 interaction. (A and B) Schemes of CP110 and CPAP illustrating the deletion mutants used in this study. “+,” interaction between CPAP and CP110; “−,” no interaction between CPAP and CP110, and “+/−,” weak interaction between CPAP and CP110. For CP110, CC1, and CC2 are the coiled-coil domains. For CPAP, CC1, and CC2 are coiled-coil domains; PN2–3, the tubulin-binding domain (Cormier et al., 2009); MBD, the MT-binding domain; and G-box, glycine-rich C-terminal domain forming an antiparallel β-sheet (Hatzopoulos et al., 2013). (C and D) Streptavidin pull-down assays with BioGFP-CP110 truncations as bait and full-length GFP-CPAP as prey. (E and F) Streptavidin pull-down assays with BioGFP-CPAP truncations as bait and full-length GFP-CP110 (E) or GFP-CP110 (581–991) (F) as prey. The assays in C–F were performed with extracts of HEK293T cells co-expressing the indicated constructs and BirA and analyzed by western blotting with anti-GFP antibodies. (G) SEC-MALS analysis of CPAP-CC1 (magenta line), CP110-CC2 (green line), and an equimolar mixture of CPAP-CC1 and CP110-CC2 (black line). (H) Scheme illustrating the mechanism for CPAP-CC1 and CP110-CC2 association. (I and J) CD spectra (I) recorded at 15°C and thermal-unfolding profiles (J) recorded by CD at 222 nm. Proteins and colors as in G. (K and L) SAXS analysis of the CPAP-CC1/CP110-CC2 heterodimer. (K) Solution X-ray scattering intensity over scattering angle from a 1:1 mixture (monomer equivalents) of CPAP-CC1 and CP110-CC2. The fit to the data yielding the interatomic distance distribution is shown with a black line. (L) Surface representation of the X-ray scattering volume of CPAP-CC1/CP110-CC2, at 32 ± 3 Å estimated precision, derived from averaging 22 particle models calculated by ab initio fit to the scattering data. Source data are available for this figure: SourceData F4.

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