Abstract
Auxilin-1 is a neuron-specific membrane-binding protein involved in a late stage of clathrin-mediated endocytosis. It recruits Hsc70, thus initiating uncoating of the clathrin-coated vesicles. Interactions of auxilin-1 with the vesicle membrane are crucial for this function and are mediated via an N-terminal PTEN-like domain. We have used multiscale molecular dynamics simulations to probe the interactions of the auxilin-1 PTEN-like domain with lipid bilayers containing differing phospholipid composition, including bilayers containing phosphatidyl inositol phosphates. Our results suggest a novel, to our knowledge, model for the auxilin/membrane encounter and subsequent interactions. Negatively charged lipids (especially PIP2) enhance binding of auxilin to lipid bilayers and facilitate its correct orientation relative to the membrane. Mutations in three basic residues (R301E/R307E/K311E) of the C2 subdomain of the PTEN-like domain perturbed its interaction with the bilayer, changing its orientation. The interaction of membrane-bound auxilin-1 PTEN-like domain with negatively charged lipid headgroups results in nanoclustering of PIP2 molecules in the adjacent bilayer leaflet.
Highlights
Clathrin-mediated endocytosis enables extracellular material, including membrane receptors and ligands, to be imported into cells through the formation of clathrin-coated vesicles [1]
Our results suggest a novel, to our knowledge, mechanism that explains how the auxilin-1 PTEN-like domain binds to membranes, and in addition reveal the formation of phosphatidyl inositol phosphate (PIP) nanoclusters in the bilayer leaflet adjacent to a bound auxilin-1 PTEN-like domain
Examination of the electrostatic potential mapped onto the surface of the auxilin-1 PTEN-like domain (Fig. 1 B) reveals three highly positively charged regions: residues 240–245 and 299–316 of the C2 domain, residues 88– 122 of the positively charged surface (PD) domain (PD; region 2), and residues 189– 212 of the PD
Summary
Clathrin-mediated endocytosis enables extracellular material, including membrane receptors and ligands, to be imported into cells through the formation of clathrin-coated vesicles [1]. Given the relatively low sequence identity, it is important to explore how the auxilin PTEN-like domain interacts with cell membranes, especially as it lacks the specific phosphatidyl inositol phosphate (PIP)-binding/ catalytic site seen in the phosphatase-domain of PTEN. Loop 3 forms a twisted b-hairpin, whereas in PTEN this loop is shorter and unstructured (Fig. S1 B) These differences are likely to be significant, especially as previous simulation studies of PTEN indicate a role of this loop in interacting with the lipid bilayer [12]. We use a multiscale approach, combining coarsegrained (CG) and atomistic (AT) MD simulations [21], to define and analyze the interactions of the PTEN-like domain of auxilin-1 with PIP-containing lipid bilayers. Our results suggest a novel, to our knowledge, mechanism that explains how the auxilin-1 PTEN-like domain binds to membranes, and in addition reveal the formation of PIP nanoclusters in the bilayer leaflet adjacent to a bound auxilin-1 PTEN-like domain
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