Structural Basis of TRPV4 N Terminus Interaction with Syndapin/PACSIN1-3 and PIP2

Abstract

Transient receptor potential (TRP) channels are polymodally regulated ion channels. TRPV4 (vanilloid 4) is sensitized by PIP₂ and desensitized by Syndapin3/PACSIN3, which bind to the structurally uncharacterized TRPV4 N terminus. We determined the nuclear magnetic resonance structure of the Syndapin3/PACSIN3 SH3 domain in complex with the TRPV4 N-terminal proline-rich region (PRR), which binds as a class I polyproline II (PPII) helix. This PPII conformation is broken by a conserved proline in a cis conformation. Beyond the PPII, we find that the proximal TRPV4 N terminus is unstructured, a feature conserved across species thus explaining the difficulties in resolving it in previous structural studies. Syndapin/PACSIN SH3 domain binding leads to rigidification of both the PRR and the adjacent PIP₂ binding site. We determined the affinities of the TRPV4 N terminus for PACSIN1, 2, and 3 SH3 domains and PIP₂ and deduce a hierarchical interaction network where Syndapin/PACSIN binding influences the PIP₂ binding site but not vice versa.

Keywords: NMR; PACSIN; PIP(2); SH3 domain; Syndapin; TRP channel; TRPV4; cis proline; class I; proline-rich region.

Figure 1:. Structural analysis of the TRPV4 N-terminus.

(A) Schematic overview of constructs with varying N-terminus length. The α-helical Ankyrin repeat domain (ARD, cyan) is preceded by a ~130 amino acid stretch harboring the PIP₂ binding domain (PBD, yellow) and the proline rich region (PRR, red) important for PACSIN SH3 domain interaction. (B) Coomassie-stained SDS-PAGE of purified chicken TRPV4 N-terminal constructs. (C) CD spectra of the entire N-terminus (N-ARD; residues 1–383), the ankyrin repeat domain with the PRR (PRR-ARD; 122–383), and the isolated Ankyrin repeat domain (ARD; 134–383). (See Fig. S1 for CD studies of human TRPV4 N-terminal constructs).

Figure 2:. Identification of the minimal TRPV4/PACSIN3 interaction sites.

(A) ¹H, ¹⁵N-HSQC NMR spectrum of PACSIN3 SH3 domain with complete backbone assignments. (B, C, D) Zoom in on peak of residue Y387 in the presence of increasing concentrations of TRPV4 N-terminal parts to show representative chemical shift changes: (B) addition of PRR-ARD (122–383) leads to chemical shift changes and line broadening due to unfavorable tumbling behavior of the 30 kDa complex; addition of (C) PRR (121–134) and (D) PBD-PRR (105–134) leads to well resolved chemical shift changes that are identical for all binding partners. Cartoon of ARD is based on (PDB: 3JXI), cartoon of PACSIN3 SH3 domain based on our NMR structure (PDB: 6F55). All interaction partners drawn to scale.

Figure 3:. Structural basis of TRPV4 interaction with PACSIN3 SH3.

(A) Solution NMR structure of TRPV4 proline rich region in complex with PACSIN3 SH3 ( see also Table 1, Fig. S4). (B) TRPV4-PRR binds to PACSIN3 SH3 domain in a class I binding mode, with N-terminal K122 interacting with the SH3 specificity pocket. P126 dips into the first proline pocket as expected for a canonical class I binding mode, but due to the cis conformation of P128, P130 rather than P129 interacts with the second proline pocket (see also Fig. S4). PACSIN1 and 2 SH3 domains share identical binding behavior (Fig. S3). (C) P128 in the TRPV4-PRR bound to the SH3 domain adopts a cis conformation while all other proline residues are in the trans conformation. (D) In the cis conformation, the proline Cδ carbon is oriented in the same direction as the backbone carboxylate of the preceding amino acid (ω=0°), in a trans conformation, they point into opposite directions (ω=180°). The cis/trans isomerization has implications for the orientation of the N- and C-terminus and thus on the position of the adjacent amino acids. (E) ¹H, ¹³C-HSQC of site specifically ¹³C-P128 labeled free (unbound) PRR shows that while P128 strongly prefers trans population in the absence of a binding partner, it has a small, detectable cis population. (F) 1D projections of the trans (blue) and cis Hγ/Cγ peaks (red) show relative populations of isomers. In the apo state, the trans state is dominant while upon addition of PACSIN1, 2 and 3 SH3 domains, the cis conformation population increases.

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.

Figure 5:. Mechanistic model of the interaction of the TRPV4-PRR with PACSIN1–3 SH3 domains.

For TRPV4 to readily react to stimuli, the channel PBD will normally be PIP₂ bound while P128 in the PRR exists mostly in the trans conformation. Because the trans/cis conformations are in exchange, PACSIN SH3 domain binding can select and stabilize P128 in the cis conformation in a “skipped class I” binding mode. This leads to TRPV4-PBD-PRR rigidification and presumably a relative reorientation of the TRPV4 N-terminus to the plasma membrane. While PIP₂ and PACSIN SH3 can form a tertiary complex in vitro, in a cellular setting the TRPV4 N-terminus reorientation and rigidification may lead to release of PIP2 and subsequent channel desensitization.