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1.
Nat Commun ; 14(1): 4165, 2023 07 13.
Article in English | MEDLINE | ID: mdl-37443299

ABSTRACT

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and the network of antagonistic regulatory elements it encodes. These modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP2 binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP2-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation.


Subject(s)
Cell Physiological Phenomena , TRPV Cation Channels , TRPV Cation Channels/metabolism , Protein Domains , Regulatory Sequences, Nucleic Acid , Lipids
2.
Chembiochem ; 24(21): e202300442, 2023 11 02.
Article in English | MEDLINE | ID: mdl-37489700

ABSTRACT

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone.


Subject(s)
Legionella pneumophila , Legionella , Legionnaires' Disease , Humans , Legionnaires' Disease/drug therapy , Legionnaires' Disease/microbiology , Tacrolimus Binding Proteins , Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/metabolism , Bacterial Proteins/metabolism , Legionella pneumophila/metabolism , Legionella/metabolism
3.
Biomol NMR Assign ; 16(2): 289-296, 2022 10.
Article in English | MEDLINE | ID: mdl-35666427

ABSTRACT

The mammalian Transient Receptor Potential Vanilloid (TRPV) channels are a family of six tetrameric ion channels localized at the plasma membrane. The group I members of the family, TRPV1 through TRPV4, are heat-activated and exhibit remarkable polymodality. The distal N-termini of group I TRPV channels contain large intrinsically disordered regions (IDRs), ranging from ~ 75 amino acids (TRPV2) to ~ 150 amino acids (TRPV4), the vast majority of which is invisible in the structural models published so far. These IDRs provide important binding sites for cytosolic partners, and their deletion is detrimental to channel activity and regulation. Recently, we reported the NMR backbone assignments of the distal TRPV4 N-terminus and noticed some discrepancies between the extent of disorder predicted solely based on protein sequence and from experimentally determined chemical shifts. Thus, for an analysis of the extent of disorder in the distal N-termini of all group I TRPV channels, we now report the NMR assignments for the human TRPV1, TRPV2 and TRPV3 IDRs.


Subject(s)
Hot Temperature , TRPV Cation Channels , Amino Acid Sequence , Amino Acids , Animals , Humans , Mammals/metabolism , Nuclear Magnetic Resonance, Biomolecular , TRPV Cation Channels/chemistry , TRPV Cation Channels/metabolism
4.
Biomol NMR Assign ; 16(2): 205-212, 2022 10.
Article in English | MEDLINE | ID: mdl-35451798

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.


Subject(s)
Chickens , TRPV Cation Channels , Amino Acids , Animals , Chickens/metabolism , Humans , Magnetic Resonance Spectroscopy , Nuclear Magnetic Resonance, Biomolecular , TRPV Cation Channels/chemistry , TRPV Cation Channels/metabolism
5.
J Mol Biol ; 433(17): 166931, 2021 08 20.
Article in English | MEDLINE | ID: mdl-33741410

ABSTRACT

The first genuine high-resolution single particle cryo-electron microscopy structure of a membrane protein determined was a transient receptor potential (TRP) ion channel, TRPV1, in 2013. This methodical breakthrough opened up a whole new world for structural biology and ion channel aficionados alike. TRP channels capture the imagination due to the sheer endless number of tasks they carry out in all aspects of animal physiology. To date, structures of at least one representative member of each of the six mammalian TRP channel subfamilies as well as of a few non-mammalian families have been determined. These structures were instrumental for a better understanding of TRP channel function and regulation. However, all of the TRP channel structures solved so far are incomplete since they miss important information about highly flexible regions found mostly in the channel N- and C-termini. These intrinsically disordered regions (IDRs) can represent between a quarter to almost half of the entire protein sequence and act as important recruitment hubs for lipids and regulatory proteins. Here, we analyze the currently available TRP channel structures with regard to the extent of these "missing" regions and compare these findings to disorder predictions. We discuss select examples of intra- and intermolecular crosstalk of TRP channel IDRs with proteins and lipids as well as the effect of splicing and post-translational modifications, to illuminate their importance for channel function and to complement the prevalently discussed structural biology of these versatile and fascinating proteins with their equally relevant 'unstructural' biology.


Subject(s)
Transient Receptor Potential Channels/metabolism , Animals , Cryoelectron Microscopy/methods , Humans , Lipids/chemistry , Mammals/metabolism , Protein Processing, Post-Translational/physiology , Proteins/metabolism , RNA Splicing/physiology
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