The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains

Abstract

How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.

Fig. 1. ATPase activities of C-terminal truncation mutants. (A) Schematics of yeast Hsp90 C-terminal truncation mutants. (B) ATPase activities of wild-type Hsp90 and C-terminal truncation mutants measured at 37°C.

Fig. 2. DMS cross-linking of Hsp90 and C-terminal truncation mutants. (A–F) SDS–PAGE gels showing cross-linking of (A) wild-type Hsp90 and C-terminal truncation mutants NC599 (B), NΔ39C551 (C), NΔ0C530 (D), NC450 (E) and NC220 (F). In all cases ‘90’ indicates the Hsp90 construct; L, DMS linker; D, ADP; N, AMP-PNP. Cross-linked species corresponding to dimers are only observed with wild-type Hsp90 and with the NC599, NΔ39C551 and NΔ0C530 C-terminal truncation mutants, only in the presence of AMP-PNP. (G) Inhibition of cross-linking of the C-terminal truncation mutant NΔ0C530 by the specific competitive inhibitors of nucleotide binding in the N-domain, geldanamycin (G) and radicicol (R). Reaction conditions are given in Materials and methods.

Fig. 3. Recruitment of p23/Sba1 to Hsp90 and C-terminal truncation mutants. (A–E) SDS–PAGE gels showing cross-linking of (A) wild-type Hsp90 and C-terminal truncation mutants NC599 (B), NΔ39C551 (C), NC450 (D) and NC220 (E). In all cases ‘90’ indicates the Hsp90 construct; 23, p23/Sba1; L, DMS linker; D, ADP; N, AMP-PNP; T, ATP. A cross-linked band higher than the Hsp90 dimer is observed with wild-type Hsp90, NC599 and NΔ39C551, only in the presence of p23/Sba1 and AMP-PNP. (F) Western blot of NC599 and p23/Sba1 cross-linking experiment. A high molecular weight band cross-reacting with anti-p23/Sba1 antisera is only observed in the presence of AMP-PNP.

Fig. 4. N-terminal interactions in single-cysteine mutants. (A) Polyacrylamide gel of purified single-cysteine mutants E7C, Q9C and E11C without added nucleotide, under non-reducing conditions and in the presence of the non-thiol reducing agent TCEP. In all three mutants, intra-dimer disulfide bonds are formed in the absence of reducing agent. (B) Excimer formation in pyrene-modified single-cysteine mutants. E11C mutant and wild-type Hsp90s were treated with pyrene (see Materials and methods), and their fluorescence emission spectra recorded. Only the cysteine mutants displayed a significant excimer signal in the region 450–500 nm, and this was substantially enhanced in the presence of AMP-PNP compared with ADP. (C) Specific excimer fluorescence signals for the E11C mutant, corrected for non-specific signal due to adsorption of the hydrophobic pyrene onto the protein surface by subtraction of the corresponding emission spectrum for the wild-type Hsp90.

Fig. 5. Opening and closing of Hsp90 molecular clamp coupled to the ATPase cycle.

Fig. 6. ATPase activities and dimerization efficiency of ts missense mutants. (A) Schematic of yeast Hsp90 primary structure with positions of missense mutations conferring ts phenotypes indicated. (B) ATPase activities of wild-type Hsp90 and ts mutants at 22, 30, 37 and 45°C. (C) Polyacrylamide gel of Hsp90 ATPase mutants T22I and T101I (6 µM) cross-linked with DMS in the presence of 10 mM AMP-PNP at 37°C for 1 h (see Materials and methods). Numbers below the lanes indicate the molar ratio of cross-linker to protein primary amine content in that reaction. At all linker concentrations the ATPase hyperactive mutant T22I shows a much higher degree of cross-linked dimers than the hypoactive T101I mutant.

Fig. 7. Hsp90 N-terminal dimer interface and lid closure. (A) Comparison of GyrB 40 kDa N-terminal fragment dimer (top) with alternative dimers in the yeast Hsp90 N-domain crystal lattice (bottom). The relative orientation of the leftmost pair (red–yellow) of Hsp90 N-domains more closely resembles that of the N-domains in GyrB than does the rightmost pair (yellow–green), originally suggested. The conformations of the ‘lid’ segments and the N-terminal strands differ between GyrB and Hsp90. AMP-PNP bound to GyrB and ADP bound to Hsp90 are shown as blue CPK models. (B) Comparison of the open conformation of the ‘lid’ segment observed in the crystal structure of yeast Hsp90 N-domain (left) with the modelled structure of the closed conformation (right). Ala107, which is exposed in the open conformation, comes close to a hydrophilic patch provided by residues Ser39, Asp40 and Asp43 in the closed conformation. Its mutation to asparagine, which is expected to stabilize this conformation, produces a dramatic increase in ATPase activity (see text). The positions of Thr22 and Thr101 are also indicated. (C) Polyacrylamide gel showing cross-linking as a function of incubation time for the C-terminal truncation mutant NΔ0C551 (wt), the A107N-NΔ0C551 double mutant (107) and the T101I-NΔ0C551 double mutant (101) cross-linked with a 15-fold molar excess of DMS, in the presence of AMP-PNP.