The three colors indicate the three different repeats. Measurement of distance between Cα R705 and Cα N651 over 450-ns simulations from the “binding” state with bound Cu(I) (BBB Cu), “binding” state without Cu(I) (BBB Apo), “extrusion” state with bound Cu(I) (EEE Cu), and “extrusion” state without Cu(I) (EEE Apo). (C) Conformational change of the CusA trimer. The distributions were taken from 3 monomers × 3 simulations, resulting in 909 data points per each analysis.
Distribution of distance between Cα R705 and Cα N651 over the last 100 ns of 450-ns simulations from the “binding” state with bound Cu(I) (BBB-Cu ), “binding” state without Cu(I) (BBB-Apo ), “extrusion” state with bound Cu(I) (EEE-Cu ) and “extrusion” state without Cu(I) (EEE-Cu ). (B) Conformational change of the CusA trimer in a modeled E. Distances between Cα atoms of R705 and N651 over 500-ns simulations from the “extrusion” state without Cu(I) (E-Apo), “extrusion” state with Cu(I) (E-Cu), “binding” state without Cu(I) (B-Apo), and “binding” state with Cu(I) (B-Cu). (A) Conformational change between bound and extruded state structure in a monomer.
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Download FIG S3, JPG file, 1.7 MB.Ĭonformational change of CusA. The shaded region indicates the standard deviation around the mean of three repeats. The secondary structure analysis was sampled every 5 ns. of 500-ns simulation of the monomeric structures with bound configuration in the presence and absence of copper (B-Cu and B-Apo) and the extruded state in the presence and absence of copper (E-Cu and E-Apo). and secondary structure retention of the monomeric structures. The secondary structure analysis was sampled every 2 ns. of the first 200-ns simulation of the trimeric cryo-EM structures with three Cu(I)-bound subunits (BBB), two Cu(I)-bound subunits, and one extrusion state (EBB), one Cu(I)-bound state (EEB), and all three subunits in the extrusion state (EEE). and secondary structure retention of the cryo-EM structures. The shaded region shows the running average every 1 ns. Download FIG S2, JPG file, 2.3 MB.Ĭα r.m.s.d. The right panel displays the proton relay network of the “resting” protomer (bright green) of the X-ray structure of apo-CusA. Likewise, the conformation of the six “extrusion” protomers are very similar to each other. The superimpositions suggest that the six “binding” protomers are very similar in conformation (left panel). The middle panel shows the comparison of the one “extrusion” protomer of CusA EBB (light green), two “extrusion” protomers of CusA EEB (pink), and three “extrusion” protomers of CusA EEE (blue). The left panel shows the overlay comparison of the one “binding” protomer of CusA EEB (light green), two “binding” protomers of CusA EBB (pink), and three binding protomers of CusA BBB (blue). In panel C, these are superimpositions of the six “binding” protomers (left panel) and six “extrusion” protomers (middle panel) from the EEB ( 7KF7), EBB ( 7KF8), EEE ( 7KF5), and BBB ( 7KF6) structures, showing the comparison of the proton relay network at different conformational states. In panel B, the distances between P54 and A754 for the “binding” (PDB ID 7KF7), “extrusion” (PDB ID 7KF5), and “resting” (PDB ID 3KO7) protomers are 7.7 Å, 8.5 Å, and 7.6 Å, respectively. The L658-L714 distance is 9.9 Å for the “resting” protomer (with the periplasmic cleft closed) of the X-ray structure of apo-CusA (PDB ID 3KO7). This distance is 9.9 Å for the “extrusion” protomer (with the periplasmic cleft closed) of the EEE structure (PDB ID 7KF5). In panel A, the distance between L658 and L714 is 20.4 Å for the “binding” protomer (with the periplasmic cleft open) of the EEB structure (PDB ID 7KF7). Different states of the CusA protomers are classified by (A) the conformation of the periplasmic cleft as measured by the distance between residues L658 (at the right side of the cleft) and L714 (at the left side of the cleft), (B) the size of the exit site of the extrusion channel as measured by the distance between residues P54 and A754, which form this exit site, and (C) the conformation of the proton relay residues, including D405, E938, and K984, in the transmembrane domain.