Fig. 3.

Conserved N540 and E543 residues are required for stable ATP-induced dimerization of Hsp70. A, Crystal structure of DnaK ATP-bound dimer (PDB code 4jne) with N537 and E540 residues highlighted in red (N540, E543 numbering in human inducible Hsp70). The NBD domain is depicted in yellow, SBDβ in marine and SBDα in green. B, Hsp70 and N540A, E543A and N540A-E543A (40 μm) were incubated without (Apo) or with ATP (0.2 mm, 20 min) at 21 °C before SEC. C, Hsp70 WT, N540A, E543A and N540A-E543A proteins (40 μm) were pre-incubated with or without ATP for 20 min before addition of chemical cross-linker (10 molar excess of DSA). The cross-linked complexes were separated by LDS-PAGE (pre-cast gradient (4–12%) gel, NuPage). The molecular weight standard is indicated. D, Native ESI-MS spectra of Hsp70 WT, N540A, E543A and N540A-E543A proteins (20 μm) were acquired after their pre-incubation without (Apo) or with ATP. The charged states corresponding to monomers and dimers are labeled with single and double dots, respectively. E, Deuteration level (exchanged deuterons, D) differences of Hsp70 WT, N540A, E543A and N540A-E543A peptides in ATP-bound and nucleotide-free state (Apo) after 1200 s (for other incubation times see supplemental Fig. S2B) incubation in deuterated buffer. Numbers at the left indicate the Hsp70 peptide fragments; schematic representation at the left shows Hsp70 domain constitution; L, interdomain linker; T, C-terminal tail. The asterisks indicate peptides covering allosterically important regions of Hsp70 molecule. Dashed lines in the graph indicate significance level of 0.35 Da (see Experimental procedures).