Fig. 2.

CHIP-K30A is intrinsically defective in E3-ligase activity. (A) Close-up of the Hsp90 binding site on CHIP extracted from the crystal structure of mCHIP dimer (protomers in shades of gray; also see Fig 1D) in complex with Hsp90 peptide (yellow sticks; PDB code 2C2L) generated using PyMOL v1.4.1. Lys³⁰ on CHIP and Asp⁷³² on Hsp90 are highlighted in blue and green respectively. (B) An AlphaScreen assay was set up (see cartoon) to measure binding dynamics of His-CHIP wt or K30A mutant with biotin-tagged Hsp70 peptide (GPTIEEVD) in solution. (C) Ubiquitination of exogenous IRF-1 in H1299 cells transiently transfected with plasmids encoding CHIP wt or K30A mutant and His-tagged ubiquitin. Immunoblots show ubiquitinated protein (His-pulldown) and total protein (Direct lysis). (D, E) In vitro ubiquitination assays were assembled using ATP, ubiquitin, UBE1, UbcH5a, untagged CHIP wt or K30A, and His-IRF-1 (D) or untagged p53 (E) as substrate. Reactions were analyzed by 4–12% NuPAGE/immunoblot. (F) Immunoblot of in vitro ubiquitination assays assembled as above except in the absence of substrate to study auto-ubiquitination of untagged CHIP wt or K30A proteins over time.