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.

Extent of intrinsic disorder and NMR chemical shift assignments of the distal N-termini from human TRPV1, TRPV2 and TRPV3 ion channels

“…NMR spectroscopy is an optimal tool to investigate highly flexible and intrinsically disordered proteins. Together with our previous study on TRPV4 (Goretzki et al 2022 ) we supplement the available structural information on group I TRPV channels with a detailed view on the extent of intrinsic disorder in their distal N-termini at atomic resolution. Interestingly, while there is some agreement with sequence-based structure predictions, the disorder determined in vitro tends to be more extensive than what is seen in silico .…”

“…Given the general lack of structural data available for the distal N-terminus in the majority of group I TRPV channel structures, many questions regarding the structural coupling of these regions to the channel pore, and the conformational propagation of peripheral ligand binding remain unanswered. We have previously described the NMR backbone assignments of the human and chicken TRPV4 IDR with 147 and 133 amino acids, respectively (Goretzki et al 2022 ). Now, to extend the structural and dynamic analysis of the IDRs to the remaining members of the human group I TRPV channels, we report the assignments of the N-terminal IDRs of the human TRPV1 (UniProtKB: Q8NER1-1, residues K2-D100, 99 amino acids), TRPV2 (UniProtKB: Q9Y5S1-1, residues T2-R73, 72 aa), and TRPV3 (UniProtKB: Q8NET8-1, residues K2-L119, 118 aa) ion channels.…”

“…Although detailed structural information on TRPV channels is available from X-ray and cryo-EM studies, in most cases the distal N-terminal region preceding the ARD escaped description at an atomic level, either because it was purposefully deleted or because it remained unresolved due to its inherent flexibility (Goretzki et al 2021 ). With a spectroscopic approach, we recently showed that the N-terminal regions of human and chicken TRPV4 channels are almost completely disordered (Goretzki et al 2018 , 2022 ). Interestingly, this was somewhat contradicted by sequence-based disorder prediction web tools that indicated the presence of a significant amount of secondary structure propensity in the TRPV4 IDRs (Goretzki et al 2022 ).…”

“…With a spectroscopic approach, we recently showed that the N-terminal regions of human and chicken TRPV4 channels are almost completely disordered (Goretzki et al 2018 , 2022 ). Interestingly, this was somewhat contradicted by sequence-based disorder prediction web tools that indicated the presence of a significant amount of secondary structure propensity in the TRPV4 IDRs (Goretzki et al 2022 ).…”

Crosstalk between regulatory elements in the disordered TRPV4 N-terminus modulates lipid-dependent channel activity

“…In some transient receptor potential (TRP) channels, IDRs make up more than half of the entire protein sequence 4 . Among the mammalian TRP vanilloid (TRPV) subfamily, TRPV4 has the largest N-terminal IDR, ranging from ~130 to ~150 amino acids in length depending on the species [4][5][6] . TRPV4 is a Ca 2+ -permeable plasma membrane channel that is widely expressed in human tissues.…”