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

Goretzki, Benedikt; Wiedemann, Christoph; McCray, Brett A.; Schaefer, Stefan L; Jansen, Jasmin; Tebbe, Frederike; Mitrovic, Sarah-Ana; Nöth, Julia; Cabezudo, Ainara Claveras; Donohue, Jack K.; Jeffries, Cy M.; Steinchen, Wieland; Stengel, Florian; Sumner, Charlotte J.; Hummer, Gerhard; Hellmich, Ute A.

doi:10.1038/s41467-023-39808-4

Cited by 18 publications

(7 citation statements)

References 93 publications

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“…The following concentrations were used: 50 μM HACE1 apo (Supplementary Data 1 ); 50 μM RAC1 Q61L ±100 μM HACE1 ∆N or 50 μM HACE1 ∆N ±100 μM RAC1 Q61L (Supplementary Data 2 ); and 25 μM of HACE1 S14E or I694D ±50 μM RAC1 Q61L (Supplementary Data 3 ). A total of 58.5 μl of D 2 O-based buffer (Supplementary Data 1 : 20 mM HEPES (pH 8.0), 150 mM NaCl, 5 mM DTT; Supplementary Data 2 and 3 : 20 mM HEPES (pH 7.8), 100 mM NaCl, 1 mM MgCl 2 , 5 mM DTT) was added to 6.5 μl of protein using a two-arm autosampler (LEAP Technologies) 101 . For additional details, see Supplementary Methods .…”

Section: Methodsmentioning

confidence: 99%

Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1

Düring,

Wolter,

Toplak

et al. 2024

Nat Struct Mol Biol

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Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of the underlying, specific E3-substrate complexes has proven challenging owing to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases (HECTs) position substrate proteins for modification. Here, we report a cryogenic electron microscopy (cryo-EM) structure of the full-length human HECT HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen–deuterium exchange mass spectrometry. Structure-based functional analyses in vitro and in cells reveal that the activity of HACE1 is stringently regulated by dimerization-induced autoinhibition. The inhibition occurs at the first step of the catalytic cycle and is thus substrate-independent. We use mechanism-based chemical crosslinking to reconstitute a complex of activated, monomeric HACE1 with its major substrate, RAC1, determine its structure by cryo-EM and validate the binding mode by solution-based analyses. Our findings explain how HACE1 achieves selectivity in ubiquitinating the active, GTP-loaded state of RAC1 and establish a framework for interpreting mutational alterations of the HACE1–RAC1 interplay in disease. More broadly, this work illuminates central unexplored aspects in the architecture, conformational dynamics, regulation and specificity of full-length HECTs.

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Section: Methodsmentioning

confidence: 99%

Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1

Düring,

Wolter,

Toplak

et al. 2024

Nat Struct Mol Biol

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“…The N-terminus of TRPV4 contains an intrinsically disordered region (IDR) of about 150 amino acids, which remains unresolved. Interestingly, in a recent study, Goretzi et al [ 125 ], have proposed that this region contains several constituents that can transiently couple and uncouple to regulate the activity of TRPV4. Such interactions were proposed to modulate the binding of lipids (i.e.…”

Section: The Bottom or Cytoplasmatic Layer Of Trpv4mentioning

confidence: 99%

“…By deleting stretches of several amino acids in the IDR and measuring the activity of the mutant TRPV4 channels, these authors proposed that there is an autoinhibitory region that acts on the PIP 2 -binding site and negatively regulates TRPV4’s activity. It was also proposed that the binding of lipids to the IDR and the PIP 2 - binding region, wields a pull force on the ARD and helps transduce mechanical forces to the rest of the TRPV4 structure [ 125 ].…”

Section: The Bottom or Cytoplasmatic Layer Of Trpv4mentioning

confidence: 99%

Recent advances on the structure and the function relationships of the TRPV4 ion channel

Sánchez-Hernández,

Benítez-Angeles,

Hernández-Vega

et al. 2024

Channels

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The members of the superfamily of Transient Receptor Potential (TRP) ion channels are physiologically important molecules that have been studied for many years and are still being intensively researched. Among the vanilloid TRP subfamily, the TRPV4 ion channel is an interesting protein due to its involvement in several essential physiological processes and in the development of various diseases. As in other proteins, changes in its function that lead to the development of pathological states, have been closely associated with modification of its regulation by different molecules, but also by the appearance of mutations which affect the structure and gating of the channel. In the last few years, some structures for the TRPV4 channel have been solved. Due to the importance of this protein in physiology, here we discuss the recent progress in determining the structure of the TRPV4 channel, which has been achieved in three species of animals ( Xenopus tropicalis , Mus musculus , and Homo sapiens ), highlighting conserved features as well as key differences among them and emphasizing the binding sites for some ligands that play crucial roles in its regulation.

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“…For the analysis of interactions between HACE1 ΔN and RAC1 Q61L, samples contained either 50 µM RAC1 +/-100 µM HACE1 or 50 µM HACE1 +/-100 µM RAC1. All samples were prepared by a two-arm autosampler (LEAP Technologies) 97 . Reactions were initiated by adding 58.5 µL of a D2Obased buffer (Dataset S1: 20 mM HEPES (pH 8.0), 150 mM NaCl, 5 mM DTT; Dataset A2: 20 mM HEPES (pH 7.8), 100 mM NaCl, 1 mM MgCl2, 5 mM DTT) to 6.5 µL of protein solution.…”

Section: Hydrogen-deuterium Exchange Mass Spectrometrymentioning

confidence: 99%

Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1

Duering,

Wolter,

Toplak

et al. 2023

Preprint

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Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. Structure determination of the underlying, specific E3-substrate complexes, however, has proven challenging due to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases position substrate proteins for modification. Here we report a cryo-EM structure of the full-length human HECT-type ligase HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry. Structure-based functional analyses in vitro and in cells reveal that the activity of HACE1 is stringently regulated by dimerization-induced autoinhibition. The inhibition occurs at the first step of the catalytic cycle and is thus substrate-independent. We employ mechanism-based chemical crosslinking to reconstitute a complex of activated, monomeric HACE1 with its major substrate, RAC1, visualize its structure by cryo-EM, and validate the binding mode by solution-based analyses. Our findings explain how HACE1 achieves selectivity in ubiquitinating the active, GTP-loaded state of RAC1 and establish a framework for interpreting mutational alterations of the HACE1-RAC1 interplay in disease. More broadly, this work illuminates central unexplored aspects in the architecture, conformational dynamics, regulation, and specificity of full-length HECT-type ligases.

show abstract

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

Cited by 18 publications

References 93 publications

Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1

Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1

Recent advances on the structure and the function relationships of the TRPV4 ion channel

Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1

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