Backbone NMR assignments of the extensive human and chicken TRPV4 N-terminal intrinsically disordered regions as important players in ion channel regulation

Abstract

Transient receptor potential (TRP) channels are important pharmacological targets due to their ability to act as sensory transducers on the organismic and cellular level, as polymodal signal integrators and because of their role in numerous diseases. However, a detailed molecular understanding of the structural dynamics of TRP channels and their integration into larger cellular signalling networks remains challenging, in part due to the systematic absence of highly dynamic regions pivotal for channel regulation from available structures. In human TRP vanilloid 4 (TRPV4), a ubiquitously expressed homotetrameric cation channel involved in temperature, osmo- and mechano-sensation and in a multitude of (patho)physiological processes, the intrinsically disordered N-terminus encompasses 150 amino acids and thus represents > 17% of the entire channel sequence. Its deletion renders the channel significantly less excitable to agonists supporting a crucial role in TRPV4 activation and regulation. For a structural understanding and a comparison of its properties across species, we determined the NMR backbone assignments of the human and chicken TRPV4 N-terminal IDRs.

Keywords: Intrinsically disordered protein; Ion channel; Regulatory domain; Structural dynamics; TRP vanilloid; Transient receptor potential.

Fig. 1

The sequence-based ODiNPred webserver predicts a significant amount of disorder in both the human (Homo sapiens, hs) and chicken (Gallus gallus, gg) TRPV4-IDR (A and B, respectively). However, several regions with low predicted disorder propensities indicate the formation of ordered structures within the TRPV4-IDR. The Z-score (upper panel) and disorder probability (lower panel) were calculated for each residue by ODiNPred (Dass et al. 2020). Residues with Z-score larger than 8 (solid line) are considered to be ordered while residues with Z-scores below 3 (dashed line) are fully disordered. Z-scores between 3 and 8 reflect transient structure formation. Regions with per-residue disorder propensities below 0.5 are shaded in light grey

Fig. 2

[¹H, ¹⁵N]-TROSY-HSQC spectra of ¹³C, ¹⁵N-labeled human (A) and chicken (B) TRPV4-IDR (147 and 133 residues, respectively) in 20 mM NaP_i, pH 4.5, 150 mM NaCl, 1 mM DTT, 0.1 mM DSS, 10% D₂O at 298 K, recorded at 800 MHz. Assigned residues are annotated in one letter amino acid code according to the human and chicken full-length TRPV4 protein sequences (UniProtKB: Q9HBA0 and A0A1D5PXA5, respectively)

Fig. 3

Chemical shift-based disorder analysis confirms that the human (A) and chicken (B) TRPV4-IDR are highly disordered throughout the entire protein sequence. (i-vii) Secondary chemical shifts calculated from the experimentally determined and predicted (using POTENCI, Nielsen and Mulder 2018) N, C’, C^α, C^β, H^α, H^β, H^N chemical shifts. (viii) Secondary structure prediction based on C^α, C^β, H^α chemical shifts using the SSP script (Marsh et al. 2006). Positive and negative values reflect α-helix and β-sheet propensities, respectively