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

Abstract

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 based on PI(4,5)P2 and PIP3 selectivity.

Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study

“…Smurf2-C2 shares 87% sequence identity with the C2 domain of Smurf1 (Figure S5), and they have similar functions and structure (Koganti et al, 2018), which make it relevant to compare them. Scott et al (2020) used MD simulations to explore interactions of InsPs with Smurf1-C2 and observed a canonical top binding mode, in line with our results. Moreover, Smurf1-C2 has been crystallized with negatively charged sulfates bound, also on the top of the C2 domain (PDB: 3PYC; Figure S5).…”

“…There have been a number of MD studies of the interaction of Ca 2+ -dependent C2 domains with membranes, revealing how Ca 2+ ions mediate interactions between the protein and anionic lipids such as phosphatidylserine (PS) and phosphatidylinositol phosphates (PIPs) ( Alwarawrah and Wereszczynski, 2017 ; Banci et al., 2002 ; Chon et al., 2015 ; Jaud et al., 2007 ; Lai et al., 2010 ; Manna et al., 2008 ; Michaeli et al., 2017 ; Vermaas and Tajkhorshid, 2017 ). However, there have not been many simulation studies of the interactions of Ca 2+ -independent C2 domains with membranes (see, e.g., Alnaas et al., 2021 ; Scott et al., 2020 ) other than for PTEN ( Shenoy et al., 2012a ; Treece et al., 2020 ), despite the growing recognition of the importance of such interactions in a number of cellular processes ( Stahelin et al., 2014 ). Here, we use coarse-grained molecular dynamics (CG-MD) ( Ingólfsson et al., 2014 ; Marrink and Tieleman, 2013 ) to examine and compare the interactions of six different Ca 2+ -independent C2 domains with lipid bilayers: C2 domains from KIBRA (PDB: 6FJD ), PI3KC2α (PDB: 6BU0 ), RIM2 (PDB: 2BWQ ), PTEN (PDB: 1D5R ), Smurf2 (PDB: 2JQZ ), and SHIP2 (PDB: 5OKM ).…”

Overexpression of TC2N is associated with poor prognosis in gastric cancer

“…Previous studies have reported the essential roles of protein containing the C2 domain in endocytosis, cellular metabolism and cancer 29 - 31 . TC2N expression is also associated with the prognosis and development of human lung cancer and breast cancer 16 - 18 .…”

Overexpression of TC2N is associated with poor prognosis in gastric cancer

“…Previous studies have reported the essential roles of protein containing the C2 domain in endocytosis, cellular metabolism and cancer [29][30][31]. TC2N expression is also associated with the prognosis and development of human lung cancer and breast cancer [16][17][18].…”