The role of lysine palmitoylation/myristoylation in the function of the TEAD transcription factors

The TEAD transcription factors are the most downstream elements of the Hippo pathway. Their transcriptional activity is modulated by different regulator proteins and by the palmitoylation/myristoylation of a specific cysteine residue. In this report, we show that a conserved lysine present in these transcription factors can also be acylated, probably following the intramolecular transfer of the acyl moiety from the cysteine. Using Scalloped (Sd), the Drosophila homolog of human TEAD, as a model, we designed a mutant protein (Glu352Gln Sd ) that is predominantly acylated on the lysine (Lys350 Sd ). This protein binds in vitro to the three Sd regulators-Yki, Vg and Tgi-with a similar affinity as the wild type Sd, but it has a significantly higher thermal stability than Sd acylated on the cysteine. This mutant was also introduced in the endogenous locus of the sd gene in Drosophila using CRISPR/Cas9. Homozygous mutants reach adulthood, do not present obvious morphological defects and the mutant protein has both the same level of expression and localization as wild type Sd. This reveals that this mutant protein is both functional and able to control cell growth in a similar fashion as wild type Sd. Therefore, enhancing the lysine acylation of Sd has no detrimental effect on the Hippo pathway. However, we did observe a slight but significant increase of wing size in flies homozygous for the mutant protein suggesting that a higher acylation of the lysine affects the activity of the Hippo pathway. Altogether, our findings indicate that TEAD/Sd can be acylated either on a cysteine or on a lysine, and suggest that these two different forms may have similar properties in cells

The Structural Basis of Calcium Dependent Inactivation of the Transient Receptor Potential Vanilloid 5 Channel.

The Transient Receptor Potential Vanilloid Channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca2+-binding protein Calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterisation of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while conferring conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with, and support the novel model for Ca2+/CaM-dependent inactivation of TRPV channels as proposed by Bate et al. (Biochemistry, 2018, in press)

The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties

The epithelial Ca2+ channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca2+ reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-terminal fragment of the channels (de Groot et al. in Mol Cell Biol 31:2845–2853, 12). Here, we investigate this binding in detail and find significant differences between TRPV5 and TRPV6. We also identify and characterize in vitro four other CaM binding fragments of TRPV5/6, which likely are also involved in TRPV5/6 channel regulation. The five CaM binding sites display diversity in binding modes, binding stoichiometries and binding affinities, which may fine-tune the response of the channels to varying Ca2+-concentrations

Regulation of TRPV5 channels by Calmodulin

The epithelial selective Ca²⁺ channel transient receptor potential vanilloid 5 channel (TRPV5) constitutes the apical entry gate for active Ca2+ reabsorption in the kidney. The activity of TRPV5 and, therefore, Ca²⁺ influx, is tightly regulated by various hormonal stimuli mediated by interactions with numerous intracellular binding partners. Previously, it was shown that the Ca²⁺-sensor Calmodulin (CaM) is directly involved in the Ca²⁺-induced inactivation of TRPV5; however, the structural basis of this mechanism remained unclear. A series of putative CaM-binding sites was identified in the TRPV5 monomer and it was shown by electrophysiology that the C-terminal distal binding site is essential for the inactivation of the channel. This thesis reports on the investigation of molecular and structural aspects of the interaction between the TRPV5 C-terminus and CaM. Using high-resolution NMR spectroscopy together with a set of complimentary methods, the CaM:TRPV5 complexes were studied in detail and the distinct roles of the CaM N- and C- domains were demonstrated. The structure of a CaM mutant, mimicking a low Ca²⁺ state, in complex with the TRPV5 peptide was determined de novo by NMR spectroscopy. Also, the interaction between the proximal C-terminal region of TRPV5 and membrane phospholipids was studied. The combined data provide the mechanistic basis for a new model of the Ca²⁺/CaM-dependent TRPV5 inactivation

The Structural Basis of Calcium-Dependent Inactivation of the Transient Receptor Potential Vanilloid 5 Channel

Accession Codes Chemical shifts and restraints were submitted to the BMRB (accession number 34161), and the ensemble of 20 conformers were submitted to the wwPDB (accession number 5OEO).The file associated with this record is under embargo until 12 months after publication, in accordance with the publisher's self-archiving policy. The full text may be available through the publisher links provided above.The transient receptor potential vanilloid channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca 2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca 2+ binding protein calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterization of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca 2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while it confers conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with and support the novel model for Ca 2+ /CaM-dependent inactivation of TRPV channels as proposed by Bate and co-workers [ Bate, N., et al. (2018) Biochemistry, (57), DOI: 10.1021/acs.biochem.7b01286 ].G.W.V. acknowledges funding during various stages of this project by the Dutch Organization of Scientific Research (NWO; 700.55.443 and 700.57.101), BBSRC (BB/J007897/1), and MRC (MR/L000555/1 and MR/P00038X/1).Peer-reviewedPost-prin

A new perspective on the evolution of the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding and in vivo function. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors

1H, 13C, 15N resonance assignment of human YAP 50–171 fragment

Yes associated protein (YAP) is an intrinsically disordered protein that plays a major role in the Hippo pathway, regulating organ size, cell proliferation, apoptosis, and is associated with cancer development. Therefore, the binding between YAP and TEAD is an interesting target for cancer therapy. The TEAD binding domain of YAP was mapped to protein residues 50–171. To obtain further structural insights into this 12 kDa segment of YAP, we report a backbone and a partial sidechain assignment of recombinant YAP 50–171.© The Author(s) 201

Structural analysis of calmodulin binding to ion channels demonstrates the role of its plasticity in regulation.

Contains fulltext : 123933.pdf (publisher's version ) (Closed access)The Ca2+-binding protein calmodulin (CaM) is a well-known regulator of ion-channel activity. Consequently, the Protein Data Bank contains many structures of CaM in complex with different fragments of ion channels that together display a variety of binding modes. In addition to the canonical interaction, in which CaM engages its target with both its domains, many of the ion-channel-CaM complexes demonstrate alternative non-canonical binding modes that depend on the target and experimental conditions. Based on these findings, several mechanisms of ion-channel regulation by CaM have been proposed, all exploiting its plasticity and flexibility in interacting with its targets. In this review, we focus on complexes of CaM with either the voltage-gated calcium channels; the voltage-gated sodium channels or the small conductance calcium-activated potassium channels, for which both structural and functional data are available. For each channel, the functional relevance of these structural data and possible mechanism of calcium-dependent (in)activation and/or facilitation are discussed in detail