Fig. 4.

CIRBP-RG/RGG region and the PY-NLS of FUS use overlapping binding sites on TNPO1. (A) ¹H-¹³C TROSY-HSQC spectrum of perdeuterated ²H-¹⁵N-¹³C-methyl-IM–labeled TNPO1 at 100 µM without (black) or with addition of one equivalent of CIRBP^RGG (orange; Left) or FUS^PY (magenta; Right). The peaks specifically affected by CIRBP^RGG or FUS^PY binding are surrounded in red, and the peaks affected by both CIRBP^RGG and FUS^PY are surrounded in black. The location of Ile and Met amino acids can be visualized on the 3D X-ray structure of TNPO1 (Protein Data Bank [PDB] ID code 2QMR) (45) with black spheres. (B) ¹H-¹⁵N TROSY-HSQC spectrum of perdeuterated ²H-¹⁵N-¹³C–labeled TNPO1 at 50 µM without (black) or with addition of one equivalent of CIRBP^RGG (orange; Left) or FUS^PY (magenta; Right). (C) ¹H-¹⁵N HSQC spectrum of ¹⁵N-labeled TNPO1^loop at 50 µM with (orange) or without (black) addition of one equivalent of CIRBP^RGG. The chemical shift perturbation (CSP) of the ¹H-¹⁵N HSQC TNPO1 loop cross-peaks between the CIRBP^RGG free and bound forms is shown in a bar plot, with the TNPO1^loop amino acids strongly affected (CSP > 0.07) by CIRBP^RGG binding colored in orange and highlighted in bold in the TNPO1^loop primary amino acid sequence. A representative cartoon is associated with each NMR spectrum with the unlabeled, NMR-invisible, protein colored gray and the labeled, NMR-visible, partner colored black (unbound), orange (CIRBP^RGG-bound), or magenta (FUS^PY-bound), respectively. (D) Electrostatic surface potential representation of TNPO1 based on its 3D structure solved in complex with FUS^PY (PDB ID code 4FQ3) using pymol 2.3.1.