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. 2019 Jun 12;10:2574. doi: 10.1038/s41467-019-10463-y

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Fig. 8 — Enhanced Hsp90–Y313E–Aha1 interaction depends on Aha1-C binding to Hsp90-M domain. a Phosphomimetic substitution of Y313 in one protomer of Hsp90 is sufficient to stimulate Aha1 association. HA or FLAG tagged Hsp90, wild-type or Y313E, were co-expressed in 293A cells and sequentially immunoprecipitated with anti-HA and anti-FLAG antibody-linked resins. The first round of IP was performed with anti-HA antibody-linked resin; bound proteins were eluted with HA peptide and then loaded onto anti-FLAG antibody-linked resin. Proteins purified from the second round of IP were eluted with SDS-sample buffer and subjected to western blot analysis. Indicated proteins were detected with specific antibodies. b Hsp90 interaction with Aha1-N is abrogated by mutation in Hsp90-M while interaction with Aha1-C is compromised by mutation in both Hsp90-N and Hsp90-M. FLAG-tagged Aha1-N or Aha1-C was co-expressed with HA-tagged Hsp90, wild-type or with the indicated mutations. Aha1 domains were immunoprecipitated with mouse anti-FLAG antibody-linked resin, and Hsp90 was detected by western blot with rat anti-HA antibody. c FLAG or HA tagged Hsp90, wild-type or mutants, were co-expressed in 293A cells and sequentially immunoprecipitated and analyzed as in panel a. Y313E-stimulated association of full-length Aha1 with Hsp90 requires phosphomimetic mutation of Y313 on only one protomer, but requires ATP binding to Hsp90-N on both protomers, since D93A mutation on either protomer negates the impact of Y313E. Aha1 binding to Hsp90-Y313E is also disrupted by W320A mutation in cis (on the same protomer containing Y313E), but no disruption occurs when W320 mutation occurs in trans to Y313E. Source data for this figure are provided as a Source Data File