TRPV4 is a cell surface-expressed calcium-permeable cation channel that mediates cell-specific effects on cellular morphology and function. Dominant missense mutations of TRPV4 cause distinct, ...tissue-specific diseases, but the pathogenic mechanisms are unknown. Mutations causing peripheral neuropathy localize to the intracellular N-terminal domain whereas skeletal dysplasia mutations are in multiple domains. Using an unbiased screen, we identified the cytoskeletal remodeling GTPase RhoA as a TRPV4 interactor. TRPV4-RhoA binding occurs via the TRPV4 N-terminal domain, resulting in suppression of TRPV4 channel activity, inhibition of RhoA activation, and extension of neurites in vitro. Neuropathy but not skeletal dysplasia mutations disrupt TRPV4-RhoA binding and cytoskeletal outgrowth. However, inhibition of RhoA restores neurite length in vitro and in a fly model of TRPV4 neuropathy. Together these results identify RhoA as a critical mediator of TRPV4-induced cell structure changes and suggest that disruption of TRPV4-RhoA binding may contribute to tissue-specific toxicity of TRPV4 neuropathy mutations.
Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins ...through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider's dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.
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 PIP
binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP
-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.
Stimulator of interferon genes (STING) is a central component of the cytosolic nucleic acids sensing pathway and as such master regulator of the type I interferon response. Due to its critical role ...in physiology and its' involvement in a variety of diseases, STING has been a focus for drug discovery. Targeted protein degradation (TPD) has emerged as a promising pharmacology for targeting previously considered undruggable proteins by hijacking the cellular ubiquitin proteasome system (UPS) with small molecules. Here, we identify AK59 as a STING degrader leveraging HERC4, a HECT-domain E3 ligase. Additionally, our data reveals that AK59 is effective on the common pathological STING mutations, suggesting a potential clinical application of this mechanism. Thus, these findings introduce HERC4 to the fields of TPD and of compound-induced degradation of STING, suggesting potential therapeutic applications.
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.
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.
Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A ...crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from
Euprosthenops australis
, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from
E. australis
as a prerequisite for obtaining further insights into protein structure and dynamics.
Objective
Distinct dominant mutations in the calcium‐permeable ion channel TRPV4 (transient receptor potential vanilloid 4) typically cause nonoverlapping diseases of either the neuromuscular or ...skeletal systems. However, accumulating evidence suggests that some patients develop mixed phenotypes that include elements of both neuromuscular and skeletal disease. We sought to define the genetic and clinical features of these patients.
Methods
We report a 2‐year‐old with a novel R616G mutation in TRPV4 with a severe neuropathy phenotype and bilateral vocal cord paralysis. Interestingly, a different substitution at the same residue, R616Q, has been reported in families with isolated skeletal dysplasia. To gain insight into clinical features and potential genetic determinants of mixed phenotypes, we perform in‐depth analysis of previously reported patients along with functional and structural assessment of selected mutations.
Results
We describe a wide range of neuromuscular and skeletal manifestations and highlight specific mutations that are more frequently associated with overlap syndromes. We find that mutations causing severe, mixed phenotypes have an earlier age of onset and result in more marked elevations of intracellular calcium, increased cytotoxicity, and reduced sensitivity to TRPV4 antagonism. Structural analysis of the two mutations with the most dramatic gain of ion channel function suggests that these mutants likely cause constitutive channel opening through disruption of the TRPV4 S5 transmembrane domain.
Interpretation
These findings demonstrate that the degree of baseline calcium elevation correlates with development of mixed phenotypes and sensitivity to pharmacologic channel inhibition, observations that will be critical for the design of future clinical trials for TRPV4 channelopathies.
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•TRP channel intrinsically disordered regions (IDRs) can present up to half of the channel sequence.•Analyses of PDB entries show that unresolved regions coincide with predicted ...disorder propensity.•TRP IDRs affect channel function, regulation and interaction with lipids and proteins.•Posttranslational modifications in TRP IDRs are discussed focusing on TRPML and TRPP channels.•IDRs need to be integrated into structural framework of TRP gating and regulation.
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.
The intrinsically disordered protein MYC belongs to the family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors (TFs). In complex with its cognate binding partner MAX, MYC ...preferentially binds to E-Box promotor sequences where it controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. Intramolecular regulation of MYC:MAX has not yet been investigated in detail. In this work, we use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map interactions between the disordered MAX N-terminus and the MYC:MAX DNA binding domain (DBD). We find that this binding event is mainly driven by electrostatic interactions and that it is competitive with DNA binding. Using NMR spectroscopy and Surface Plasmon Resonance (SPR), we demonstrate that the MAX N-terminus serves to accelerate DNA binding kinetics of MYC:MAX and MAX:MAX dimers, while it simultaneously provides specificity for E-Box DNA. We also establish that these effects are further enhanced by Casein Kinase 2-mediated phosphorylation of two serine residues in the MAX N-terminus. Our work provides new insights how bHLH-LZ TFs are regulated by intramolecular interactions between disordered regions and the folded DNA binding domain.
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