Figure 4. Ubiquitin receptors Rpn13 and S5a bind K48-linked diubiquitin simultaneously.

A) ¹H, ¹³C HSQC experiments recorded on ¹³C labeled S5a (196–306) alone (black) or with equimolar K48-linked diubiquitin (green) or K48-linked diubiquitin and Rpn13 (1–150) (blue) reveal P276 (left panel) and V222 (right panel) to interact with diubiquitin when Rpn13 is present. B) ¹H, ¹⁵N HSQC experiments on ¹⁵N labeled Rpn13 (1–150) alone (black) and with monoubiquitin (orange), K48-linked diubiquitin (red) or K48-linked diubiquitin and S5a (196–306) (blue) reveals L73 to shift to its diubiquitin-bound state when S5a is present. K48-linked diubiquitin with its (C) proximal or (D) distal subunit ¹⁵N labeled alone (black) or with Rpn13 (1–150) (red), S5a (196–306) (green), or both of these receptors (blue) indicates shifting that mimics Rpn13 binding to the proximal subunit, but S5a binding to the distal one, as shown for F45 and Q62 of the proximal subunit and I44 and I61 of the distal subunit. Additional data is provided in Supplementary Figure 6. (E) Model illustrating Rpn13 bound to K48-linked diubiquitin’s proximal subunit and S5a’s UIMs interacting dynamically with the distal one.