Interactions of the ion channel TRPV4 with regulatory lipids and proteins - An (un)structural study
Date issued
Authors
Editors
Journal Title
Journal ISSN
Volume Title
Publisher
License
Abstract
Transient receptor potential vanilloid 4 (TRPV4) is a polymodal eukaryotic cation channel that acts as a transducer of mechanical and osmotic stress, temperature, and pain in multiple human tissues. TRPV4 is regulated by a remarkably diverse spectrum of interaction partners to maintain cellular homeostasis. Perturbances in these regulatory pathways, e.g. through mutations in TRPV4, have been implicated with various sensory and motor neuropathies. For TRPV4 to react to temperature and osmotic stress stimuli, the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) needs to interact with the TRPV4 phosphoinositide binding domain (PBD). The channel’s sensitivity for temperature and osmotic stress decreases when the F-BAR domain protein Pacsin3 binds with its SH3 domain to a proline-rich region (PRR) in the proximity of the PBD. PRR and the PBD are both located in a ~150 residue region preceding an ankyrin repeat domain (ARD) in the cytosolic TRPV4 N-terminus. This region is missing in the currently available high-resolution structure of a full-length TRPV4 channel. Sequence analyses predict it to be an intrinsically disordered region (IDR). Due to the lack of structural information, the molecular mechanism underlying the PIP2- and Pacsin3-mediated regulation of TRPV4 remains enigmatic.
The results of this thesis provide detailed insights into the structural dynamics of a hitherto uncharacterized region of TRPV4 and how it interacts with channel modulators. A structural and dynamic coupling be-tween ARD and IDR emerges as the mechanistic basis for sensing ligand-binding in the IDR in the remaining channel regions. The binding partners PIP2 and Pacsin3 may induce counteracting IDR conformations with opposing effects on the ARD/IDR interaction. PIP2 binding to the PBD leads to an IDR association with the plasma membrane, thereby sensitizing TRPV4. In contrast, Pacsin3 binding to the PRR stabilizes a conformation that tilts the IDR into the cytosol. An exclusive IDR-interaction of PIP2 or Pacsin3 appears as an explanation of the antagonistic relationship between both channel modulators. Beyond the PIP2/Pacsin3 mediated regulation of TRPV4, the ARD/IDR communication may link other events in the IDR such as post-translational modifications or channelopathy-mutations to the functional state of the channel. The ARD/IDR communication, thus, forms the basis for a novel mechanism that may explain how the intrinsically unfolded N-terminus in a physiologically important TRP vanilloid member can regulate its channel activity.
Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy combined with hydrogen/deuterium-exchange (HDX-MS) and chemical cross-linking mass spectrometry (XL-MS) re-veal that the entirely disordered IDR is structurally and dynamically coupled to the ARD. In contrast to the expectations based on high-resolution structures of the isolated ARD, this a-helical domain is a comparably labile protein in solution and seems to adopt transiently unfolded states. In the context of the NTD, the ARD becomes structurally and dynamically stabilized through an interaction with the IDR. Strikingly, the PBD seems to contribute to the IDR-ARD communication and may thus link the dynamics in the ARD to a PBD/PIP2 interaction and potentially to Pacsin3 binding to the PRR. NMR and fluorescence spectroscopy revealed that the PBD is generally capable of binding anionic lipids and that regions beyond the PBD interact with PIP2 and other anionic lipids, even though with a lower affinity. This observation indicates that the IDR will be associated with the plasma membrane in the sensitized state of TRPV4, therefore enabling the PBD to bind to a PIP2 lipid. NMR spectroscopy was further used to characterize the Pacsin3 SH3 domain’s interaction with the TRPV4 PRR and the putative cross-talk with the PBD/PIP2 interaction. Competitive binding experiments show that the Pacsin3 SH3 domain and PIP2 can bind to their binding sites in the IDR simultaneously. A solution NMR structure of the Pacsin3 SH3 domain in complex with the TRPV4 PRR reveals that Pacsin3 binding requires a proline trans to cis isomerization in the PRR. This isomerization reorients the PRR and presumably rearranges the preceding IDR. Pacsin3 may antagonize the effects of PIP2 on TRPV4 by stabilizing an IDR conformation where the PBD loses contact with a PIP2 molecule in the plasma membrane. Structural analyses of full-length Pacsin3 via SAXS, X-ray crystallography, NMR, and XL-MS suggest that an F-BAR/SH3 domain interaction maintains Pacsin3 in a compact, putatively auto-inhibited, conformation in the absence of a substrate. The binding of the TRPV4 PRR releases the SH3 domain from the F-BAR domain and presumably activates Pacsin3, thus leading to F-BAR domain-induced membrane curvate. Thus, the regulation of TRPV4 by PIP2 and Pacsin3 appears to be governed by structural rearrangements in the participating protein interaction partners.