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Molecular Analysis of Membrane Targeting by the C2 Domain of the E3 Ubiquitin Ligase Smurf1

1
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
2
Department of Medicinal Chemistry and Molecular Pharmacology and the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
*
Author to whom correspondence should be addressed.
Biomolecules 2020, 10(2), 229; https://doi.org/10.3390/biom10020229
Received: 6 January 2020 / Revised: 28 January 2020 / Accepted: 29 January 2020 / Published: 4 February 2020
(This article belongs to the Section Biochemistry)
SMAD ubiquitination regulatory factor 1 (Smurf1) is a Nedd4 family E3 ubiquitin ligase that regulates cell motility, polarity and TGFβ signaling. Smurf1 contains an N-terminal protein kinase C conserved 2 (C2) domain that targets cell membranes and is required for interactions with membrane-localized substrates such as RhoA. Here, we investigated the lipid-binding mechanism of Smurf1 C2, revealing a general affinity for anionic membranes in addition to a selective affinity for phosphoinositides (PIPs). We found that Smurf1 C2 localizes not only to the plasma membrane but also to negatively charged intracellular sites, acting as an anionic charge sensor and selective PIP-binding domain. Site-directed mutagenesis combined with docking/molecular dynamics simulations revealed that the Smurf1 C2 domain loop region primarily interacts with PIPs and cell membranes, as opposed to the β-surface cationic patch employed by other C2 domains. By depleting PIPs from the inner leaflet of the plasma membrane, we found that PIP binding is necessary for plasma membrane localization. Finally, we used a Smurf1 cellular ubiquitination assay to show that the amount of ubiquitin at the plasma membrane interface depends on the lipid-binding properties of Smurf1. This study shows the mechanism by which Smurf1 C2 targets membrane-based substrates and reveals a novel interaction for non-calcium-dependent C2 domains and membrane lipids. View Full-Text
Keywords: C2 domain; E3 ubiquitin ligase; lipid binding; phosphoinositide; plasma membrane; Smurf1; ubiquitin C2 domain; E3 ubiquitin ligase; lipid binding; phosphoinositide; plasma membrane; Smurf1; ubiquitin
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Scott, J.L.; Frick, C.T.; Johnson, K.A.; Liu, H.; Yong, S.S.; Varney, A.G.; Wiest, O.; Stahelin, R.V. Molecular Analysis of Membrane Targeting by the C2 Domain of the E3 Ubiquitin Ligase Smurf1. Biomolecules 2020, 10, 229.

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