Figure 4: Interactions between TRPV4 N-terminus, PACSIN3 SH3 domain and PIP₂.

(A) Chemical shift differences of TRPV4-PBD-PRR (bottom) and TRPV4-PBD^AAWAA-PRR (top) free and bound states with PACSIN3 SH3 (red and light grey traces) and PIP₂ (yellow and dark grey traces). PIP₂ only leads to chemical shift changes in the previously determined PIP₂ binding domain (Garcia-Elias et al., 2013), while PACSIN3 SH3 domain has effects on both the PRR and the PBD. For PBD^AAWAA-PRR, mostly the same residues are affected by PACSIN3 SH3 binding, while PIP₂ binding leads to line broadening in the linker region (empty boxes) and only very slight chemical shifts in the PBD. (B) {¹H},¹⁵N-HetNOE values of TRPV4-PBD-PRR backbone amides. Lower values are indicative of higher flexibility. (C) Position of the three phosphate groups, P1, P4 and P5 in PI(4,5)P₂. (D) ³¹P titration curves of PIP₂ P4 and P5 with PBD-PRR (blue), PBD^AAWAA-PRR (green) and PRR (red) derived from ³¹P chemical shift changes: (E-G) ³¹P NMR spectra of PIP₂ phosphate groups with (E) PBD-PRR; (F) PBD^AAWAA-PRR and (G) PRR. (H) Effect of adding PIP₂ and PBD-PRR to ¹⁵N-PACSIN3 SH3 domain in different orders:Peak corresponding to PACSIN3 SH3 Y387 is shown in the 2D ¹H, ¹⁵N-spectrum and as a slice through the nitrogen dimension to illustrate line width (left, grey)Upon addition of PIP₂ to the SH3 domain, no chemical shift differences nor line broadening is observed, thus showing that lipid and SH3 domain do not interact (bottom, orange). In contrast, addition of PBD-PRR to ¹⁵N-PACSIN3 SH3 shows a chemical shift and concomitant line broadening indicative of interaction between SH3 domain and peptide (top, blue). In a second step, the respective missing binding partner (PIP₂ in upper and PBD-PRR in lower path) is added. In both cases, identical final chemical shift positions and broad line widths are observed (right) showing that PACSIN3 SH3, PIP₂ and TRPV4-PBD-PRR form the same tripartite complex regardless of order of binding partner addition.