Figure 1. Labeling sites and putative conformational switch.
Schematic of spin-labeled sites in the extracellular, intracellular and NBD regions of TM287/288 in (A) the inward-facing apo crystal structure (PDB: 4Q4H) and in (B) the outward-facing homology model based on the Sav1866 crystal structure (PDB: 2HYD). TM287 is colored in cyan and TM288 in pink. (C–E) Simulations of the distance distribution probabilities for the six spin-labeled double mutants in the IF (cyan) and OF (magenta) states represented in panels A and B. The ambient temperature MTSL rotamer library in MMM2015 was used. Comparison with the experimental data and with a previous version of the MTSL library are presented in Figure 1—figure supplement 1.
Figure 1—figure supplement 1. Comparison between simulated and experimental distance distributions.
Simulations of the distance distribution probability for the six spin-labeled pairs in the IF (cyan) and OF (magenta) states performed with MMM2015 with the rotamer library at ambient temperature (dotted lines as in Figure 1) and with the rotamer library of MMM2013 simulated at 175 K (dashed lines). The experimental distance distributions on nucleotide-free (apo, black solid line in upper plots) and ATP-Vi-Mg (black, solid line in lower plots) states are taken from Figure 2. Transparent cyan and magenta rectangles outline the range of experimental distances characteristic of the IF and OF conformations, respectively.
Figure 1—figure supplement 2. Hoechst 33342 stimulated ATPase activities of wildtype TM287/288 and spin-labeled mutants reconstituted into proteoliposomes.
Stimulation of ATPase hydrolysis of wildtype TM287/288 and six spin-labeled mutants reconstituted into proteoliposomes was determined in the absence (basal activity) and in the presence of 50 µM, 100 µM and 150 µM Hoechst 33342 at 50°C. Data were normalized to the basal ATPase activity of reconstituted wildtype TM287/288. The error bars are standard deviations of three technical replicates.