Abstract
Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are essential players in the dynamics of intracellular compartments. The BAR domain is an evolutionarily conserved dimeric module characterized by a crescent-shaped structure whose intrinsic curvature, flexibility, and ability to assemble into highly ordered oligomers contribute to inducing the curvature of target membranes. Endophilins, diverging into A and B subgroups, are BAR and SH3 domain-containing proteins. They exert activities in membrane dynamic processes such as endocytosis, autophagy, mitochondrial dynamics, and permeabilization during apoptosis. Here, we report on the involvement of the third α-helix of the endophilin A BAR sequence in dimerization and identify leucine 215 as a key residue within a network of hydrophobic interactions stabilizing the entire BAR dimer interface. With the combination of N-terminal truncation retaining the high dimerization capacity of the third α-helices of endophilin A and leucine 215 substitution by aspartate (L215D), we demonstrate the essential role of BAR sequence-mediated dimerization on SH3 domain partnership. In comparison with wild type, full-length endophilin A2 heterodimers with one protomer bearing the L215D substitution exhibit very significant changes in membrane binding and shaping activities as well as a dramatic decrease of SH3 domain partnership. This suggests that subtle changes in the conformation and/or rigidity of the BAR domain impact both the control of membrane curvature and downstream binding to effectors. Finally, we show that expression, in mammalian cells, of endophilin A2 bearing the L215D substitution impairs the endocytic recycling of transferrin receptors.
Highlights
The BAR domain is a dimeric module controlling membrane curvature
The binding of full-length recombinant Endophilin A1 (EA1) to poly-L-proline, of low efficiency, requires the SH3 domain because the SH3-deleted (⌬SH3) form does not interact with poly-Lproline (Fig. 1A and see supplemental Fig. 1 for an overview of all mutant and tagged forms used in this study)
While purifying a series of N-terminally truncated forms of EA1 encompassing the three helices of the BAR sequence via binding to immobilized poly-L-proline, we observed that the form devoid of the first ␣-helix was immobilized with low efficiency but that proteins lacking the two N-terminal helices (EA1⌬(1–175) and EA1⌬(1–200)) could bind much more efficiently (Fig. 1A)
Summary
The BAR domain is a dimeric module controlling membrane curvature. Results: We identify leucine 215 involved in stabilizing the dimer interface and characterize the incidence of its substitution in SH3-mediated partnership in endophilins. Functional Relationship of BAR/SH3 Domains of Endophilin proposed to induce membrane curvature, stabilize curvature driven by other mechanical forces, or sense curvature as a signal for selective recruitment on a given target membrane as well as for the shape-driven attachment to other factors that may act as downstream effectors Their activity on membranes was proposed to rely on oligomerization properties that propagate positive curvature to shape tubular structures, as exemplified by self-assembly of F-BAR modules into a helical coat [4]. We identify leucine 215 as playing a central role in stabilizing hydrophobic interactions between BAR protomers and characterize the impaired activities of the BAR domain of endophilin A2 bearing the L215D substitution in membrane deformation, partnership of the SH3 domain, and endocytic recycling of the transferrin receptor
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