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. 2022 Feb 9;298(3):101697. doi: 10.1016/j.jbc.2022.101697

Figure 8.

Figure 8

Pseudophosphorylation of the Hsp70 C terminus inhibits CHIP binding but has no effect on DnaJB4.A, saturation binding fluorescence polarization experiments comparing WT or phosphomimetic T636E Hsp72 fluorescent tracer binding to CHIP (left) or DnaJB4 (right). Increasing concentrations of CHIP or DnaJB4 were incubated with Hsp72 fluorescent tracer for 30 min at room temperature. The results are the average of four replicates, and error bars represent SD. The Kd values for each condition are expressed as mean with a 95% confidence interval. B, comparison between the binding modes of Hsp70’s EEVD motif interacting with the CHIP or DnaJB4. The CHIP tetratricopeptide repeat (TPR) (green) orients the Hsp70 EEVD motif in a hooked or bent conformation, where it makes interactions with the P5 Ile, P2 Val, P1 Asp, and C-terminal carboxylate. Conversely, CTD I of DnaJB4 (blue) binds the Hsp70 EEVD in a linear orientation, with the P5 Ile and C-terminal carboxylate making important contacts. Residues important for binding to each cochaperone are highlighted on the crystal structures (CHIP TPR-Hsp70 EEVD: PDB 6EFK (35), DnaJB1 CTD I-Hsp70 EEVD: PDB 3AGY (36)).