Figure 6.

PRE mapping of Bro1–hyperphosphorylated$\mathbf{PR}{\mathbf{D}}_{\mathbf{703}–\mathbf{868}}^{\mathbf{Strep}}$interactions.A, schematic of $\mathrm{P}\mathrm{R}{\mathrm{D}}_{\mathrm{703}–\mathrm{868}}^{\mathrm{S}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{p}}$; vertical lines indicate the location of the four independent conjugation sites and of the P801G mutation (green). The location of phosphorylated tyrosine residues is shown. B, intermolecular PREs observed on ²H/¹⁵N-labeled Bro1 (200 μM) arising from MTSL label conjugated to hyperphosphorylated $\mathrm{P}\mathrm{R}{\mathrm{D}}_{\mathrm{703}–\mathrm{868}}^{\mathrm{S}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{p}}$ at four specific sites; Bro1 to $\mathrm{P}\mathrm{R}{\mathrm{D}}_{\mathrm{703}–\mathrm{868}}^{\mathrm{S}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{p}}$ molar ratio = 1:1. Conjugation of MTSL at site C813 produced a noticeable intermolecular PRE; residues with intermolecular PREs above the background are labeled and indicated by purple bars. PREs too large (>60 s⁻¹) to be accurately measured are plotted as 60 s⁻¹. The remaining three conjugation sites did not generate large intermolecular PREs; a few exceptions are labeled. C, ribbon representation of Bro1; residues that exhibit large PREs (≥25 s⁻¹) with spin label at site C813 are depicted as purple spheres; residues that were completely broadened out are marked with asterisks. Y319 is shown in a stick representation. D, molecular surface of Bro1 (in the same orientation as C) color coded according to electrostatic potential; ±5 kT with blue, positive; white, neutral; and red, negative. PRD, proline-rich domain; PRE, paramagnetic relaxation enhancement; MTSL, (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate.