Fig. 1: Positive charge at position 380 in the M domain of Hsp90 is critical for closed clamp formation.
a Cartoon showing the PET experimental setup. Molecules in the open conformation (left) have high fluorescence. Molecules in the closed clamp conformation (right) have low fluorescence due to quenching of the fluorophore in the N domain by an engineered tryptophan (W) in the M domain. b The response of wild type Hsp82 (left) and Hsp82E33A to AMPPNP (red), ATP-γ-S (blue) and ATP (black) in the PET assay. Nucleotides were added to the proteins at T = 10 min. c Cartoon depicting the motion (blue arrows) of the ATP γ-phosphate in the open state (left) and bound to R380 in the closed clamp (right). d Like in c. except with AMPPNP. The γ imidophosphate of AMPPNP is structurally predisposed to contact R380 (compare to position of γ-phosphate in c.) and thus forms the closed clamp more readily than ATP (compare black arrows to c.). e The response of Hsp82R380A (left) and Hsp82R380K (right) to AMPPNP (red), ATP-γ-S (blue) and ATP (black) in the PET assay. f ATPase rates of Hsp82E33A, Hsp82R380A, and Hsp82R380K relative to wild type Hsp82. Bars are the average rates of at least three reactions (each replicate is shown as a circle) and error bars are the standard deviation. Asterisks indicate significant difference from wild type (P < 0.0001 by one-way ANOVA). g In vivo Hsp90 functional assay (See “Methods”). Cells expressing Hsp82R380A were inviable while those expressing Hsp82R380K grew like wild type on plates containing FOA. h Cells expressing Hsp82R380K had a similar growth rate as cells expressing wild type Hsp82 (not significant by one-way ANOVA). Bars are the average growth rates of three biological replicates (each replicate is shown as a circle) and error bars are the standard deviation. i Cells expressing Hsp82R380K were able to chaperone kinase clients since they grew like wild type on calcofluor white (CFW, see text). Cells expressing Hsp82E33A are shown as a CFW sensitive control. Lines in the images indicate where portions were cropped from different plates of identical composition.
