PMC

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 , ).

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).

A) Crystal structure of TRAP1 rotated 75° from view in . Highlighted regions are the distinct MD:CTD interfaces generated by the helix swap of protomers A and B highlighted in . 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).

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 )

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 )

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 ).

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 , ) 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 )