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Unfolding Mechanisms and Conformational Stability of the Dimeric Endophilin N-BAR Domain.

ABSTRACT: Endophilin, which is a member of the Bin-amphiphysin-Rvs (BAR) domain protein superfamily, contains a homodimeric N-BAR domain of a characteristic crescent shape. The N-BAR domain comprises a six-helix bundle and is known to sense and generate membrane curvature. Here, we characterize aspects of the unfolding mechanism of the endophilin A1 N-BAR domain during thermal denaturation and examine factors that influence the thermal stability of this domain. Far-UV circular dichroism (CD) spectroscopy was applied to monitor changes in the secondary structure above room temperature. The protein's conformational changes were further characterized through Foerster resonance energy transfer and cross-linking experiments at varying temperatures. Our results indicate that thermal unfolding of the endophilin N-BAR is (minimally) a two-step process, with a dimeric intermediate that displays partial helicity loss. Furthermore, a thermal shift assay and temperature-dependent CD were applied to compare the unfolding processes of several truncated versions of endophilin. The melting temperature of the N-BAR domain decreased when we deleted either the N-terminal H0 helix or the unstructured linker of endophilin. This result suggests that these intrinsically disordered domains may play a role in structurally stabilizing the functional N-BAR domain in vivo. Finally, we show that single-site mutations can also compromise endophilin's thermal stability.

SUBMITTER: Jin R

PROVIDER: S-EPMC8374900 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundEndophilin is a cytoplasmic protein with an important function in clathrin-dependent endocytosis at synapses and elsewhere. Endophilin has a BAR (Bin/Amphiphysin/Rvs-homology) domain, which is implicated in the sensing and induction of membrane curvature. Previous structure-function studies of the endophilin-A BAR domain have almost exclusively been made in reduced systems, either in vitro or ex vivo in cultured cells. To extend and complement this work, we have analyzed the role played by the structural features of the endophilin-A BAR domain in Drosophila in vivo.Methodology/principal findingsThe study is based on genetic rescue of endophilin-A (endoA) null mutants with wild type or mutated endoA transgenes. We evaluated the viability of the rescuants, the locomotor behavior in adult flies and the neurotransmission at the larval neuromuscular junction. Whereas mutating the endophilin BAR domain clearly affected adult flies, larval endophilin function was surprisingly resistant to mutagenesis. Previous reports have stressed the importance of a central appendage on the convex BAR surface, which forms a hydrophobic ridge able to directly insert into the lipid bilayer. We found that the charge-negative substitution A66D, which targets the hydrophobic ridge and was reported to completely disrupt the ability of endophilin-BAR to tubulate liposomes in vitro, rescued viability and neurotransmission with the same efficiency as wild type endoA transgenes, even in adults. A similar discrepancy was found for the hydrophilic substitutions A63S/A66S and A63S/A66S/M70Q. The A66W mutation, which introduces a bulky hydrophobic side chain and induces massive vesiculation of liposomes in vitro, strongly impeded eye development, even in presence of the endogenous endoA gene. Substantial residual function was observed in larvae rescued with the EndoA(Arf) transgene, which encodes a form of endophilin-A that completely lacks the central appendage. Whereas a mutation (D151P) designed to increase the BAR curvature was functional, another mutation (P143A, DeltaLEN) designed to decrease the curvature was not.Conclusions/significanceOur results provide novel insight into the structure/function relationship of the endophilin-A BAR domain in vivo, especially with relation to synaptic function.

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Unfolding Mechanisms and Conformational Stability of the Dimeric Endophilin N-BAR Domain.

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