Preprint Article Version 1 This version is not peer-reviewed

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

Version 1 : Received: 6 January 2020 / Approved: 8 January 2020 / Online: 8 January 2020 (04:34:28 CET)

How to cite: Scott, J.; Frick, C.; Johnson, K.; Liu, H.; Yong, S.; Varney, A.; Wiest, O.; Stahelin, R. Molecular Analysis of Membrane Targeting by the C2 Domain of the E3 Ubiquitin Ligase Smurf1. Preprints 2020, 2020010062 (doi: 10.20944/preprints202001.0062.v1). Scott, J.; Frick, C.; Johnson, K.; Liu, H.; Yong, S.; Varney, A.; Wiest, O.; Stahelin, R. Molecular Analysis of Membrane Targeting by the C2 Domain of the E3 Ubiquitin Ligase Smurf1. Preprints 2020, 2020010062 (doi: 10.20944/preprints202001.0062.v1).

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.

Subject Areas

C2 domain; E3 ubiquitin ligase; lipid binding; phosphoinositide; plasma membrane; smurf1; ubiquitin

Comments (0)

We encourage comments and feedback from a broad range of readers. See criteria for comments and our diversity statement.

Leave a public comment
Send a private comment to the author(s)
Views 0
Downloads 0
Comments 0
Metrics 0


×
Alerts
Notify me about updates to this article or when a peer-reviewed version is published.