Structural basis for RNA slicing by a plant Argonaute.

Argonaute (AGO) proteins use small RNAs to recognize transcripts targeted for silencing in plants and animals. Many AGOs cleave target RNAs using an endoribonuclease activity termed 'slicing'. Slicing by DNA-guided prokaryotic AGOs has been studied in detail, but structural insights into RNA-guided slicing by eukaryotic AGOs are lacking.

Inhibition of Autophagy by a Small Molecule through Covalent Modification of the LC3 Protein.

The autophagic ubiquitin-like protein LC3 functions through interactions with LC3-interaction regions (LIRs) of other autophagy proteins, including autophagy receptors, which stands out as a promising protein-protein interaction (PPI) target for the intervention of autophagy. Post-translational modifications like acetylation of Lys49 on the LIR-interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering with the interface. Through screening covalent compounds, we discovered a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49.

Selenomethionine as an expressible handle for bioconjugations.

Site-selective chemical bioconjugation reactions are enabling tools for the chemical biologist. Guided by a careful study of the selenomethionine (SeM) benzylation, we have refined the reaction to meet the requirements of practical protein bioconjugation. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine.

Structure, lipid scrambling activity and role in autophagosome formation of ATG9A.

De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer.

ATG2A transfers lipids between membranes in vitro.

ATG2 is a rod-shaped membrane-tethering protein suggested to mediate the endoplasmic reticulum (ER)-phagophore association and the expansion of the phagophore. We recently demonstrated that human ATG2A transfers lipids between membranes in vitro, which led us to propose a model that the phagophore expands upon the transfer of lipids from the ER by ATG2. Here, we summarize our findings and arising issues that need to be addressed to establish the mechanism of phagophore expansion.

The autophagic membrane tether ATG2A transfers lipids between membranes.

An enigmatic step in de novo formation of the autophagosome membrane compartment is the expansion of the precursor membrane phagophore, which requires the acquisition of lipids to serve as building blocks. Autophagy-related 2 (ATG2), the rod-shaped protein that tethers phosphatidylinositol 3-phosphate (PI3P)-enriched phagophores to the endoplasmic reticulum (ER), is suggested to be essential for phagophore expansion, but the underlying mechanism remains unclear. Here, we demonstrate that human ATG2A is a lipid transfer protein.

The rod-shaped ATG2A-WIPI4 complex tethers membranes in vitro.

The autophagosome precursor membrane, termed the "isolation membrane" or "phagophore," emerges adjacent to a PI3P-enriched transient subdomain of the ER called the "omegasome," thereafter expanding to engulf cytoplasmic content. Uncovering the molecular events that occur in the vicinity of the omegasome during phagophore biogenesis is imperative for understanding the mechanisms involved in this critical step of the autophagy pathway. We recently characterized the ATG2A-WIPI4 complex, one of the factors that localize to the omegasome and play a critical role in mediating phagophore expansion.

Structural Studies of Mammalian Autophagy Lipidation Complex.

Members of the autophagy-related protein 8 (Atg8) family of ubiquitin-like proteins (ublps), including mammalian LC3 and GABARAP proteins, play crucial roles in autophagosome biogenesis, as well as selective autophagy. Upon induction of autophagy, the autophagic ublps are covalently attached to a phosphatidylethanolamine (PE) molecule of the autophagosomal membrane. This unique lipid conjugation of the autophagic ublps, which is essential for their functions, occurs in a ubiquitination-like reaction cascade consisting of the E1 enzyme ATG7, the E2 ATG3, and the E3 ATG12~ATG5-ATG16L1 complex (~denotes a covalent linkage).

Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex.

Autophagy is an enigmatic cellular process in which double-membrane compartments, called "autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4.

Identification and characterization of the linear region of ATG3 that interacts with ATG7 in higher eukaryotes.

Transfer of GABARAP thioester from the E1 ATG7 to the E2 ATG3 requires the interaction between the N-terminal domain of ATG7 and the flexible region (FR) of ATG3. This interaction has been visualized in the yeast Atg7-Atg3 complex crystal structure, but remains to be defined in higher eukaryotes. Here, our NMR data precisely define the region of the FR of human ATG3 that interacts with ATG7 (RIA7) and demonstrate RIA7 partially overlaps with the E3-interacting region, explaining how the E1-E2 and E2-E3 interactions are mutually exclusive.

Structural insights into E2-E3 interaction for LC3 lipidation.

The members of the LC3/Atg8 family of proteins are covalently attached to phagophore and autophagosomal membranes. At the last step of the LC3 lipidation cascade, LC3 is transferred from the E2 enzyme ATG3 to phosphatidylethanolamine (PE). This transfer is stimulated by the ATG12-ATG5-ATG16L1 E3 complex, but the mechanism is not fully understood.

Structural basis of ATG3 recognition by the autophagic ubiquitin-like protein ATG12.

The autophagic ubiquitin-like protein (ublp) autophagy-related (ATG)12 is a component of the ATG12∼ATG5-ATG16L1 E3 complex that promotes lipid conjugation of members of the LC3 ublp family. A role of ATG12 in the E3 complex is to recruit the E2 enzyme ATG3. Here we report the identification of the ATG12 binding sequence in the flexible region of human ATG3 and the crystal structure of the minimal E3 complexed with the identified binding fragment of ATG3.

Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy.

The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase-like activity which facilitates the lipidation of members of the LC3 family. The crystal structure of the human ATG12~ATG5 conjugate bound to the N-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues. ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjugation junction, form a continuous surface patch.

Crystal structure of the Formin mDia1 in autoinhibited conformation.

Background: Formin proteins utilize a conserved formin homology 2 (FH2) domain to nucleate new actin filaments. In mammalian diaphanous-related formins (DRFs) the FH2 domain is inhibited through an unknown mechanism by intramolecular binding of the diaphanous autoinhibitory domain (DAD) and the diaphanous inhibitory domain (DID).

Methodology/principal Findings: Here we report the crystal structure of a complex between DID and FH2-DAD fragments of the mammalian DRF, mDia1 (mammalian diaphanous 1 also called Drf1 or p140mDia).

A novel mechanism of actin filament processive capping by formin: solution of the rotation paradox.

The FH2 domains of formin family proteins act as processive cappers of actin filaments. Previously suggested stair-stepping mechanisms of processive capping imply that a formin cap rotates persistently in one direction with respect to the filament. This challenges the formin-mediated mechanism of intracellular cable formation.

Structural basis of Rho GTPase-mediated activation of the formin mDia1.

Diaphanous-related formins (DRFs) regulate dynamics of unbranched actin filaments during cell contraction and cytokinesis. DRFs are autoinhibited through intramolecular binding of a Diaphanous autoinhibitory domain (DAD) to a conserved N-terminal regulatory element. Autoinhibition is relieved through binding of the GTPase RhoA to the N-terminal element.

Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain.

The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus.

Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular Cdc42 signaling.

The temporal and spatial control of Rho GTPase signaling pathways is a central issue in understanding the molecular mechanisms that generate complex cellular movements. The Rho protein Cdc42 induces a significant conformational change in its downstream effector, the Wiskott-Aldrich syndrome protein (WASP). On the basis of this conformational change, we have created a series of single-molecule sensors for both active Cdc42 and Cdc42 guanine nucleotide exchange factors (GEFs) that utilize fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins.