Structural asymmetry in the closed state of mitochondrial Hsp90 (TRAP1) supports a two-step ATP hydrolysis mechanism

Abstract

While structural symmetry is a prevailing feature of homo-oligomeric proteins, asymmetry provides unique mechanistic opportunities. We present the crystal structure of full-length TRAP1, the mitochondrial Hsp90 molecular chaperone, in a catalytically active closed state. The TRAP1 homodimer adopts a distinct, asymmetric conformation, where one protomer is reconfigured via a helix swap at the middle:C-terminal domain (MD:CTD) interface. This interface plays a critical role in client binding. Solution methods validate the asymmetry and show extension to Hsp90 homologs. Point mutations that disrupt unique contacts at each MD:CTD interface reduce catalytic activity and substrate binding and demonstrate that each protomer needs access to both conformations. Crystallographic data on a dimeric NTD:MD fragment suggests that asymmetry arises from strain induced by simultaneous NTD and CTD dimerization. The observed asymmetry provides the potential for an additional step in the ATPase cycle, allowing sequential ATP hydrolysis steps to drive both client remodeling and client release.

Figure 1. Crystal structure of full-length TRAP1 in an asymmetric closed state

A) Full-length TRAP1 homodimer from D. rerio. Protomer B (orange) is similar to the p23-stabilized yHsp90 structure, while protomer A (blue) makes novel contacts between the MD and CTD. Residues known to bind clients in this region are shown in red. Also visible is an N-terminal extension that exchanges between protomers. Cobalt atoms (pink) help stabilize crystal contacts. B) TRAP1 domain boundaries (left), map of the helical positions within a single protomer (right). N to C-terminal progression is indicated by a transition from blue to gray. C) Comparison of TRAP1 structure to the closed state of yHsp90 (gray) (RMSD = 3.5Å). (See also Movie S1)

Figure 2. N-terminal strand extension regulates TRAP1 activity

A) The surface representation of protomer A highlights the electrostatic charge distribution of the NTD and the extensive NTD-strand swap (1,484 Ų/monomer) made with each neighboring protomer. B) The salt bridge between H87 and E157 is displayed with the electron density map calculated using experimental phase restraints. C) ATPase activities of WT TRAP1, Δstrap, and H87 or E157 mutations indicate that disruption of strap contacts leads to a significant acceleration of ATPase activity. (Error bars are propagated standard deviations).

Figure 3. Novel asymmetry revealed in the TRAP1 dimer

A) Protomers from TRAP1 and yHsp90 full-length structures are aligned at the LMD, highlighting asymmetry, between TRAP1 domains. RMSD values highlight differences between the LMD and SMD versus the entire MD; the direction and degree of rotation calculated between subdomains is indicated in the inset. B) Domain differences between Trap1 protomers are illustrated with a thicker diameter and the color yellow highlighting regions of higher variability (higher RMSD). C) View of the NTD:MD interface with protomers aligned at the NTD illustrates that asymmetry starts near R417. D) Global alignment yHsp90 and TRAP1 protomers shows significant differences in overall RMSD values. The zoomed panel shows helix swapping at the unique interface formed at the MD:CTD interface. (see also Movie S2)

Figure 4. Solution methods support a conserved asymmetric closed state

A) SAXS P(r) curves show that addition of ATP analogs to TRAP1 homologs results in a characteristic shift towards a more compact conformation. Overlaying the nucleotide bound SAXS curves reveals little difference in the closed conformation between homologs. B) SAXS curves of closed state hTRAP1, zTRAP1 from panel A, and bHsp90 from Krukenberg et al. fit using a linear combination of apo data and theoretical scattering for the zTRAP1 crystal structure (asymmetric), or a closed state model of zTRAP1 in the yHsp90 conformation (symmetric). Residuals below clearly show that asymmetric structure is best fit. (see also Table S1, Figure S1) C) DEER probe design shown on protomers A and B aligned at the CTD with a predicted distance change of 14 Å. D) Background corrected and normalized time-domain DEER data (black) fit by Tikhonov regularization (red) are shown on the left. Calculated distance distributions for closed state hTRAP1 (right) obtained after Tikhonov regularization. Two major peaks are observed in the presence of ADP-BeF supporting an asymmetric closed state in solution. Colored arrows show calculated distances from protomer A (blue) or B (orange). (see also Figure S2)

Figure 5. Structure based MD:CTD interface mutations impair ATPase activity

A) Crystal structure of TRAP1 rotated 75° from view in Figure 1A. Highlighted regions are the distinct MD:CTD interfaces generated by the helix swap of protomers A and B highlighted in Figure 3. B) Relative k_obs of zTRAP1 and hTRAP1 with single point mutations designed to disrupt unique contacts at the MD:CTD interfaces. The equivalent drop in activity establishes conservation of asymmetric interfaces between homologs. (Error bars are propagated standard deviations).

Figure 6. NTD:MD crystal structure and implications for a strained closed state

A) Crystal structure of a symmetric closed NTD:MD: AMPPNP TRAP1 dimer formed by CTD cleavage during crystallization that retains NTD dimerization. Cleavage of the CTD resulted from destabilization of the MD:CTD interface by point mutations. B) Overlay of the NTD:MD state with the full-length TRAP1 crystal structure shows that in absence of strain imposed by simultaneous NTD/CTD dimerization, TRAP1 protomers relax outward to a symmetric conformation. (see also Figure S6/S7, Movie S3).

Figure 7. New model for the conformational cycle of Hsp90

In the absence of nucleotide, Hsp90 exists in an equilibrium of states with varying open conformations. Upon ATP binding the chaperone shifts to an asymmetric closed conformation that is significantly strained leading to buckling of the MD:CTD interface (client binding residues in red, transparency for visualization). Upon hydrolysis of one ATP, strain is relieved and the MD:CTD interface is re-arranged perhaps forming a symmetric state reminiscent of the yHsp90 conformation. This conformation can be stabilized by dual binding of co-chaperone p23 (purple) at the NTD stalling the progression of the cycle. Upon hydrolysis of the second ATP, the ADP state is transiently formed and ADP release resets the cycle to the apo state equilibrium. (Protomer arms are colored as in Figure 1).