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 for non-calcium-dependent C2 domains and membrane lipids.

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 S2), and they have similar functions and structure (Koganti et al, 2018), which make it relevant to compare them. Scott et al used MD simulations to explore interactions of InsPs with Smurf1-C2 and they saw a canonical top-binding mode (Scott et al, 2020), in line with our results. Moreover, Smurf1-C2 has been crystallised with negatively charged sulphates bound, also in the top of the C2 domain (PDB ID 3PYC; SI Figure S2).…”

“…However, there have not been many simulation studies of the interactions of Ca 2+ -independent C2 domains with membranes (see e.g. (Scott et al, 2020)) other than for PTEN (S. Shenoy et al, 2012; Treece et al, 2020) despite the growing recognition of the importance of such interactions in a number of cellular processes (Stahelin et al, 2014).…”

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted October 30, 2020. ; https://doi.org/10.1101/2020.10.30.361964 doi: bioRxiv preprint C2_main v26MS.docx 30-Oct-20 there have not been many simulation studies of the interactions of Ca 2+ -independent C2 domains with membranes (see e.g. (Scott et al, 2020)) other than for PTEN (S. 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 ID 6FJD), PI3KC2α (PDB ID 6BU0), RIM2 (PDB ID 2BWQ), PTEN (PDB ID 1D5R), Smurf2 (PDB ID 50KM) and SHIP2 (PDB ID 2JQZ)).…”

Specific interactions of peripheral membrane proteins with lipids: what can molecular simulations show us?

“…Whilst not necessarily as robust as structural analysis, this approach is able to form a solid basis for downstream analysis using MD. This approach has, e.g., been applied to investigate the PIP-binding sites of Smurf1-C2 [ 65 ], as well as interactions of the E. coli heat-labile enterotoxin with the blood group A pentasaccharide [ 42 ]. The high-throughput nature of the approach also makes docking of other molecules, such as potential therapeutic drugs, feasible, as shown with an expansive analysis of alkaloid binding to Ctx [ 66 ].…”

As a matter of fat: Emerging roles of lipid‐sensitive E3 ubiquitin ligases

“…SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) is a lipidresponsive ligase involved in signalling pathways at the plasma membrane and may also change metabolic responses through lipid signalling. [47] It is a peripheral membrane protein and member of the NEDD4 subfamily of HECT E3 ubiquitin ligases that responds to changes in plasma membrane lipids. [48,49] SMURF1 regulates bone morphogenetic protein (BMP) activity by targeting BMP receptors type I (BMPR) and TGF-β receptor type I (TβRI) for proteasome-mediated degradation.…”