Figure 5. The asymmetry is flipped in the ATP/ADP state as revealed by DEER on hemi-hydrolyzed (ATP/ADP) heterodimers.

The cartoons depict hTRAP1 heterodimers (one protomer colored by domains and the other in gray; NTD, blue; MD, green; CTD, brown) with spin-labels (red circles) and the relevant nucleotide state (D or T, for ADP or ATP, respectively) as well as the R402A mutation (ADP state mimic; magenta star). The SpyCatcher-SpyTag is shown attached to the CTD tails. (A) +/+heterodimers (green line) partition roughly equally between the buckled (left, 22 Å) and straight (right, 41 Å) conformations. (B) Spin-labels on the opposite (trans) protomer of +/R402A heterodimers (blue line) show that nearly all molecules are buckled on the ATP arm. (C) +/R402A heterodimers (orange line) carrying spin-labels on the same (cis) protomer as the R402A mutation, showing that the protomer prefers the straight conformation. (D) Crystal structure of +/R417A heterodimeric zTrap1 with the SpyCatcher-SpyTag fusion in the asymmetric closed state showing buckled (blue) and straight (orange) protomers. The dashed line indicates disordered residues. Insets show that the γ-phosphate-sensing R417 is only present on the buckled arm. Phases come from a model having Ala on both protomers. 2Fo-Fc density (gray mesh) and Fo-Fc difference map (green mesh) around the position of the asymmetric R417A mutation. Strong positive density (green mesh) on the difference map is observed only at the buckled protomer.

DOI: http://dx.doi.org/10.7554/eLife.25235.010

Figure 5—figure supplement 1. Interatomic distance distribution, P(r), from SAXS experiments of heterodimeric +/R402A human TRAP1.

The heterodimer is fully capable of closing, shown as overall reduction in the width and maximum dimension of the P(r) distribution upon incubation with an ATP analog (ADP-BeF, teal). The black curve is the P(r) of the apo dimer.

DOI: http://dx.doi.org/10.7554/eLife.25235.011

Figure 5—figure supplement 2. Crystal packing interactions for the heterodimeric (+/R417A) zTRAP1 fused to the SpyCatcher-Tag domains.

A pair of symmetry mates is colored purple and teal whose SpyCatcher-Tag domains pack against each other (black inset, a close-up of the dotted circle). The SpyCatcher and SpyTag belonging to one dimer are colored purple and red, respectively. The corresponding SpyCatcher-SpyTag symmetry mate is colored teal and yellow, respectively.

DOI: http://dx.doi.org/10.7554/eLife.25235.012