Fig. 3: E372K rescues R32A and E33A growth defects in vivo and restores closed clamp formation by ATP-γ-S in vitro.

a Hsp90 functional assay with the indicated inviable nucleotide binding pocket mutants (left column) and the same but combined with E372K. The E372K mutation rescued R32A but not the others. Growth of one replicate of G123A is an escape event and not true complementation, see “Methods”. Both images were cropped from the same plate. b Cells expressing Hsp82^R32A,E372K were viable but slow growing (left) and CFW sensitive (right). c The response of Hsp82^R32A,E372K mutant to AMPPNP (red), ATP-γ-S (blue), and ATP (black) in the PET assay. Compare blue solid and dashed (Hsp82^R32A plus ATP-γ-S, reproduced from Fig. 2a) lines. d The response of Hsp82^E33A,E372K mutant to AMPPNP (red), ATP-γ-S (blue), and ATP (black) in the PET assay. Compare blue solid to dashed (Hsp82^E33A plus ATP-γ-S, reproduced from Fig. 1b) lines. For c, d nucleotides were added to the proteins at T = 10 min.