Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery

Abstract

Hsp90 is a molecular chaperone essential for the activation and assembly of many key eukaryotic signalling and regulatory proteins. Hsp90 is assisted and regulated by co-chaperones that participate in an ordered series of dynamic multiprotein complexes, linked to Hsp90s conformationally coupled ATPase cycle. The co-chaperones Aha1 and Hch1 bind to Hsp90 and stimulate its ATPase activity. Biochemical analysis shows that this activity is dependent on the N-terminal domain of Aha1, which interacts with the central segment of Hsp90. The structural basis for this interaction is revealed by the crystal structure of the N-terminal domain (1-153) of Aha1 (equivalent to the whole of Hch1) in complex with the middle segment of Hsp90 (273-530). Structural analysis and mutagenesis show that binding of N-Aha1 promotes a conformational switch in the middle-segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N-terminal nucleotide-binding domain of the chaperone.

Figure 1

Aha1 N-terminal domain structure. (A) Secondary structure cartoon of the N-terminal domain of Aha1 (residues 1–153) rainbow coloured (blue → red) from the N- to C-terminus of the domain. The molecule has an overall cylindrical structure. (B) Alignment of the sequences for the N-terminal domains of representative Aha1 homologues from budding yeast (Sc), fission yeast (Sp), plants (At), flies (Dm), worms (Ce) and humans (Hs), and the entire sequence of the yeast Hch1 (Sc). Secondary structural elements are marked as in (A). The strongly conserved Trp/Asn-rich and basic motifs are highlighted in blue; regions involved in contacts with Hsp90 (see below) are highlighted in yellow.

Figure 2

Hsp90–Aha1 lattice contacts imitating client-protein interactions. (A) Two consecutive Hsp90 middle-segment molecules in different asymmetric units in the high-resolution crystal form, both make contact with an Aha1 N-domain molecule. The highlighted interactions between the right-hand M-Hsp90 molecule and N-Aha1 are lattice contacts that do not occur in the second crystal form. However, these involve residues in Hsp90 that have been implicated in interactions with client proteins (Sato et al, 2000; Meyer et al, 2003), and suggest that this contact may reflect the kind of interaction Hsp90 makes with client proteins. (B) Detailed view of the interaction between the exposed hydrophobic side chain of Trp 300 in the putative client-binding site of Hsp90, and a shallow hydrophobic recess on the outer face of Aha1.

Figure 3

Hsp90–Aha1 complex. (A) The middle segment of Hsp90 contacts the Aha1 N-terminal domain over the entire length of both molecules, with interactions involving all three subdomains of M-Hsp90. Apart from a core hydrophobic interaction (see (B)), the interface involves many ion pairs and direct and solvent bridged polar interactions. Solvent molecules bound at the interface are show as green spheres. (B) Detailed view of the interface between the first αβα domain of M-Hsp90 and N-Aha1, centred on the hydrophobic interaction between Ile 64, Leu 66 and Phe 100 from Aha1 and Leu 315, Ile 388 and Val 391 from Hsp90. Above this, the two proteins interact via a ‘ladder' of ion pairs formed between Asp 53, Asp 101, Asp 68 and Glu 97 from Aha1, and lysines 387, 390, 394 and 398 from Hsp90. Side chains of Hsp90 residues are shown in white, and those of Aha1 in cyan.

Figure 4

Aha1 proximity to the nucleotide-binding domain. (A) Docking of the crystal structure of the M-Hsp90–N-Aha1 complex onto the model for the combined N-terminal and middle segments of an Hsp90 monomer (Meyer et al, 2003) directs the C-terminus of the Aha1 N-terminal domain (rainbow coloured) towards the nucleotide-binding domain of Hsp90. The C-terminal domain of Aha1, which is not present in the smaller Hch1 homologue, could interact with the nucleotide-binding domain to further enhance ATP turnover. (B) Comparison of stimulation of yeast Hsp90 ATPase activity by wild type (wt) and Δ11-Aha1, in which the disordered N-terminal 11 residues have been deleted. Although it contains the NxNNWHW motif, which is highly conserved in Aha1 and Hch1 sequences, deletion of the N-terminal 11 residues produced only a very small decrease in the degree of activation elicited by Aha1, suggesting that it is not involved in the activation mechanism.

Figure 5

Conformational switching in the γ-phosphate-binding loop. (A) Comparison of the conformations of the γ-phosphate-binding loops (highlighted in gold) from the distantly related GHKL-family proteins GyrB, MutL and Hsp90. In the GyrB and MutL structures, where the N-terminal domain (not shown) contains a bound ATP analogue, the loops adopt an active conformation in which the catalytically essential basic residue (cyan) can contact the γ-phosphate. In uncomplexed Hsp90 in the absence of a nucleotide-bound N-terminal domain, the loop adopts a retracted structure with a short helix, in which the catalytic Arg 380 points back to the middle segment. (B) Detailed view of the retracted conformation of the catalytic loop (370–390) in Hsp90. Gln 384 and Lys 387, and the catalytically essential Arg 380 make ion-pair and polar interactions with a series of acidic residues (Glu 353, Glu 355 and Asp 356) on the overlying structure. (C) Detailed view of the catalytic loop of Hsp90 in the M-Hsp90–N-Aha1 complex. Binding of Aha1 causes the side chain of Lys 387 to move >15 Å, breaking its intramolecular ion-pair interaction with Asp 356 to hydrogen bond to the main-chain carbonyl of Leu 66 and ion pair with the side-chain carboxyl of Asp 53 of Aha1. Simultaneously, Ile 88 moves >8 Å to pack against the side chain of Leu 66 on Aha1. These movements add an extra turn to the beginning of the long helix following the catalytic loop in Hsp90, and destabilise the short helix (379–386), releasing Arg 380 from its retracted and inactive interaction with Glu 353 and Glu 355. (D, E) As (C), but from two other crystallographically independent copies of the M-Hsp90–N-Aha1 complex, showing the plasticity of the Hsp90 middle-segment catalytic loop.

Figure 6

Functional analysis of Hsp90–Aha1 interacting residues. (A) The basic RKGK motif in Aha1 forms ion pairs with acidic side chains in the catalytic loop that will stabilise the active conformation. Mutation of all these residues to alanine produces a substantial decrease in ATPase activation as judged by the maximal activation achieved at saturation, but with little decrease in affinity. (B) Asp 53 in Aha1 and Lys 387 in Hsp90 form part of the core interaction between the two proteins following a binding-induced conformational change in the 370–390 loop of Hsp90. Mutation of either of these residues to alanine, which permits the conformational change, causes a decrease in affinity but only a small decrease in ATPase activation. Charge-reversal mutations at either residue prevent adoption of the binding-induced conformation and substantially decreases activation and affinity.