Members of the Bin/amphiphysin/Rvs (BAR) domain protein superfamily are involved in membrane remodeling in various cellular pathways ranging from endocytic vesicle and T-tubule formation to cell migration and neuromorphogenesis. Membrane curvature induction and stabilization are encoded within the BAR or Fer-CIP4 homology-BAR (F-BAR) domains, α-helical coiled coils that dimerize into membrane-binding modules. BAR/F-BAR domain proteins often contain an SH3 domain, which recruits binding partners such as the oligomeric membrane-fissioning GTPase dynamin. How precisely BAR/F-BAR domain-mediated membrane deformation is regulated at the cellular level is unknown. Here we present the crystal structures of full-length syndapin 1 and its F-BAR domain. Our data show that syndapin 1 F-BAR-mediated membrane deformation is subject to autoinhibition by its SH3 domain. Release from the clamped conformation is driven by association of syndapin 1 SH3 with the proline-rich domain of dynamin 1, thereby unlocking its potent membrane-bending activity. We hypothesize that this mechanism might be commonly used to regulate BAR/F-BAR domain-induced membrane deformation and to potentially couple this process to dynamin-mediated fission. Our data thus suggest a structure-based model for SH3-mediated regulation of BAR/F-BAR domain function.

Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1–kinase complex. However, the underlying molecular mechanism remains unclear. Here we reconstitute this early step in autophagy from purified components in vitro. We find that on assembly from its cytoplasmic subcomplexes, the Atg1–kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. The scaffolding protein Atg17 targets the Atg1–kinase complex to autophagic membranes by specifically recognizing the membrane protein Atg9. This interaction is inhibited by the two regulatory subunits Atg31 and Atg29. Engagement of the Atg1–Atg13 subcomplex restores the Atg9-binding and membrane-tethering activity of Atg17. Our data help to unravel the mechanism that controls Atg17-mediated tethering of Atg9-vesicles, providing the molecular basis to understand initiation of autophagosome-biogenesis.

BAR domain superfamily proteins have emerged as central regulators of dynamic membrane remodeling, thereby playing important roles in a wide variety of cellular processes, such as organelle biogenesis, cell division, cell migration, secretion, and endocytosis. Here, we review the mechanistic and structural basis for the membrane curvature-sensing and deforming properties of BAR domain superfamily proteins. Moreover, we summarize the present state of knowledge with respect to their regulation by autoinhibitory mechanisms or posttranslational modifications, and their interactions with other proteins, in particular with GTPases, and with membrane lipids. We postulate that BAR superfamily proteins act as membrane-deforming scaffolds that spatiotemporally orchestrate membrane remodeling.

Diaryl ketones are important building blocks for synthesizing pharmaceuticals and are generally regarded as “difficult-to-reduce” ketones due to the large steric hindrance of their two bulky aromatic side chains. Alcohol dehydrogenase from Kluyveromyces polyspora (KpADH) has been identified as a robust biocatalyst due to its high conversion of diaryl ketone substrate (4-chlorophenyl)­(pyridine-2-yl)­ketone (CPMK) with a moderate R-selectivity of 82% ee. To modulate the stereoselectivity of KpADH, a “polarity scanning” strategy was proposed, in which six key residues inside and at the entrance of the substrate binding pocket were identified. After iterative combinatorial mutagenesis, variants Mu-R2 and Mu-S5 with enhanced (99.2% ee, R) and inverted (97.8% ee, S) stereoselectivity were obtained. The crystal structures of KpADH and two mutants in complex with NADPH were resolved to elucidate the evolution of enantioselective inversion. Based on MD simulation, Mu-R2–CPMKProR and Mu-S5–CPMKProS were more favorable in the formation of prereaction states. Interestingly, a quadrilateral plane formed by α-carbons of four residues (N136, V161, C237, and G214) was identified at the entrance of the substrate binding pocket of Mu-S5; this plane acts as a “polar gate” for substrates. Due to the discrepancy in charge characteristics between chlorophenyl and pyridine substituents, the pro-S orientation of CPMK is defined when it passes through the “polar gate” in Mu-S5, whereas the similar plane in wild-type is blocked by several aromatic residues. Our result paves the way for engineering stereocomplementary ADH toward bulky diaryl ketones and provides structural insight into the mechanism of stereoselective inversion.