Characterization of PROPPIN-Phosphoinositide Binding and Role of Loop 6CD in PROPPIN-Membrane Binding.

PROPPINs (ß-propellers that bind polyphosphoinositides) are a family of PtdIns3P- and PtdIns(3,5)P2-binding proteins that play an important role in autophagy. We analyzed PROPPIN-membrane binding through isothermal titration calorimetry (ITC), stopped-flow measurements, mutagenesis studies, and molecular dynamics (MD) simulations. ITC measurements showed that the yeast PROPPIN family members Atg18, Atg21, and Hsv2 bind PtdIns3P and PtdIns(3,5)P2 with high affinities in the nanomolar to low-micromolar range and have two phosphoinositide (PIP)-binding sites. Single PIP-binding site mutants have a 15- to 30-fold reduced affinity, which explains the requirement of two PIP-binding sites in PROPPINs. Hsv2 bound small unilamellar vesicles with a higher affinity than it bound large unilamellar vesicles in stopped-flow measurements. Thus, we conclude that PROPPIN membrane binding is curvature dependent. MD simulations revealed that loop 6CD is an anchor for membrane binding, as it is the region of the protein that inserts most deeply into the lipid bilayer. Mutagenesis studies showed that both hydrophobic and electrostatic interactions are required for membrane insertion of loop 6CD. We propose a model for PROPPIN-membrane binding in which PROPPINs are initially targeted to membranes through nonspecific electrostatic interactions and are then retained at the membrane through PIP binding.

Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a ß-propeller protein family.

ß-propellers that bind polyphosphoinositides (PROPPINs), a eukaryotic WD-40 motif-containing protein family, bind via their predicted ß-propeller fold the polyphosphoinositides PtdIns3P and PtdIns(3,5)P(2) using a conserved FRRG motif. PROPPINs play a key role in macroautophagy in addition to other functions. We present the 3.0-Å crystal structure of Kluyveromyces lactis Hsv2, which shares significant sequence homologies with its three Saccharomyces cerevisiae homologs Atg18, Atg21, and Hsv2. It adopts a seven-bladed ß-propeller fold with a rare nonvelcro propeller closure. Remarkably, in the crystal structure, the two arginines of the FRRG motif are part of two distinct basic pockets formed by a set of highly conserved residues. In comprehensive in vivo and in vitro studies of ScAtg18 and ScHsv2, we define within the two pockets a set of conserved residues essential for normal membrane association, phosphoinositide binding, and biological activities. Our experiments show that PROPPINs contain two individual phosphoinositide binding sites. Based on docking studies, we propose a model for phosphoinositide binding of PROPPINs.

Crystal structure of the autochaperone region from the Shigella flexneri autotransporter IcsA.

The IcsA (intracellular spread gene A) autotransporter from Shigella flexneri is a key virulence factor. We identified a stable fragment comprising residues 591 to 758, which corresponds to the autochaperone region of the IcsA passenger domain. We showed that thermal unfolding of the autochaperone region is reversible and determined its crystal structure at 2.0-Å resolution.

Crystal structure of the Rab33B/Atg16L1 effector complex.

The Atg12-Atg5/Atg16L1 complex is recruited by WIPI2b to the site of autophagosome formation. Atg16L1 is an effector of the Golgi resident GTPase Rab33B. Here we identified a minimal stable complex of murine Rab33B(30-202) Q92L and Atg16L1(153-210). Atg16L1(153-210) comprises the C-terminal part of the Atg16L1 coiled-coil domain. We have determined the crystal structure of the Rab33B Q92L/Atg16L1(153-210) effector complex at 3.47 Å resolution. This structure reveals that two Rab33B molecules bind to the diverging α-helices of the dimeric Atg16L1 coiled-coil domain. We mutated Atg16L1 and Rab33B interface residues and found that they disrupt complex formation in pull-down assays and cellular co-localization studies. The Rab33B binding site of Atg16L1 comprises 20 residues and immediately precedes the WIPI2b binding site. Rab33B mutations that abolish Atg16L binding also abrogate Rab33B association with the Golgi stacks. Atg16L1 mutants that are defective in Rab33B binding still co-localize with WIPI2b in vivo. The close proximity of the Rab33B and WIPI2b binding sites might facilitate the recruitment of Rab33B containing vesicles to provide a source of lipids during autophagosome biogenesis.

Crystal structures of substrate-free and retinoic acid-bound cyanobacterial cytochrome P450 CYP120A1.

The crystal structures of substrate-free and all-trans-retinoic acid-bound CYP120A1 from Synechocystis sp. PCC 6803 were determined at 2.4 and 2.1 A resolution, respectively, representing the first structural characterization of a cyanobacterial P450. Features of CYP120A1 not observed in other P450 structures include an aromatic ladder flanking the channel leading to the active site and a triple-glycine motif within SRS5. Using spectroscopic methods, CYP120A1 is shown to bind 13-cis-retinoic acid, 9-cis-retinoic acid, and retinal with high affinity and dissociation constants of less than 1 microM. Metabolism of retinoic acid by CYP120A1 suggests that CYP120A1 hydroxylates a variety of retinoid derivatives in vivo. On the basis of the retinoic acid-bound CYP120A1 crystal structure, we propose that either carbon 2 or the methyl groups (C16 or C17) of the beta-ionone ring are modified by CYP120A1.

Crystal structure of the human retinitis pigmentosa 2 protein and its interaction with Arl3.

The crystal structure of human retinitis pigmentosa 2 protein (RP2) was solved to 2.1 angstroms resolution. It consists of an N-terminal beta helix and a C-terminal ferredoxin-like alpha/beta domain. RP2 is functionally and structurally related to the tubulin-specific chaperone cofactor C. Seven of nine known RP2 missense mutations identified in patients are located in the beta helix domain, and most of them cluster to the hydrophobic core and are likely to destabilize the protein. Two residues, Glu138 and the catalytically important Arg118, are solvent-exposed and form a salt bridge, indicating that Glu138 might be critical for positioning Arg118 for catalysis. RP2 is a specific effector protein of Arl3. The N-terminal 34 residues and beta helix domain of RP2 are required for this interaction. The abilitities of RP2 to bind Arl3 and cause retinitis pigmentosa seem to be correlated, since both the R118H and E138G mutants show a drastically reduced affinity to Arl3.

Structure based biophysical characterization of the PROPPIN Atg18 shows Atg18 oligomerization upon membrane binding.

PROPPINs (ß-propellers that bind polyphosphoinositides) are PtdIns3P and PtdIns(3,5)P2 binding autophagy related proteins. They contain two phosphatidylinositolphosphate (PIP) binding sites and a conserved FRRG motif is essential for PIP binding. Here we present the 2.0 Å resolution crystal structure of the PROPPIN Atg18 from Pichia angusta. We designed cysteine mutants for labelling with the fluorescence dyes to probe the distances of the mutants to the membrane. These measurements support a model for PROPPIN-membrane binding, where the PROPPIN sits in a perpendicular or slightly tilted orientation on the membrane. Stopped-flow measurements suggest that initial PROPPIN-membrane binding is driven by non-specific PIP interactions. The FRRG motif then retains the protein in the membrane by binding two PIP molecules as evident by a lower dissociation rate for Atg18 in comparison with its PIP binding deficient FTTG mutant. We demonstrate that the amine-specific cross-linker Bis(sulfosuccinimidyl)suberate (BS3), which is used for protein-protein cross-linking can also be applied for cross-linking proteins and phosphatidylethanolamine (PE). Cross-linking experiments with liposome bound Atg18 yielded several PE cross-linked peptides. We also observed intermolecular cross-linked peptides, which indicated Atg18 oligomerization. FRET-based stopped-flow measurements revealed that Atg18 rapidly oligomerizes upon membrane binding while it is mainly monomeric in solution.

Analyzing Protein-Phosphoinositide Interactions with Liposome Flotation Assays.

Liposome flotation assays are a convenient tool to study protein-phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein-lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method.

Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E.

The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.

The GTPase Rab26 links synaptic vesicles to the autophagy pathway.

Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.

It takes two to tango: PROPPINs use two phosphoinositide-binding sites.

PROPPINs are a family of PtdIns3P and PtdIns(3,5)P 2-binding proteins. The crystal structure now unravels the presence of two distinct phosphoinositide-binding sites at the circumference of the seven bladed ß-propeller. Mutagenesis analysis of the binding sites shows that both are required for normal membrane association and autophagic activities. We identified a set of evolutionarily conserved basic and polar residues within both binding pockets, which are crucial for phosphoinositide binding. We expect that membrane association of PROPPINs is further stabilized by membrane insertions and interactions with other proteins.

Crystal structure of the human short coiled coil protein and insights into SCOC-FEZ1 complex formation.

The short coiled coil protein (SCOC) forms a complex with fasciculation and elongation protein zeta 1 (FEZ1). This complex is involved in autophagy regulation. We determined the crystal structure of the coiled coil domain of human SCOC at 2.7 Å resolution. SCOC forms a parallel left handed coiled coil dimer. We observed two distinct dimers in the crystal structure, which shows that SCOC is conformationally flexible. This plasticity is due to the high incidence of polar and charged residues at the core a/d-heptad positions. We prepared two double mutants, where these core residues were mutated to either leucines or valines (E93V/K97L and N125L/N132V). These mutations led to a dramatic increase in stability and change of oligomerisation state. The oligomerisation state of the mutants was characterized by multi-angle laser light scattering and native mass spectrometry measurements. The E93V/K97 mutant forms a trimer and the N125L/N132V mutant is a tetramer. We further demonstrate that SCOC forms a stable homogeneous complex with the coiled coil domain of FEZ1. SCOC dimerization and the SCOC surface residue R117 are important for this interaction.

Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods.

We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P 2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.

Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment.

Synaptic-vesicle exocytosis is mediated by the vesicular Ca(2+) sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca(2+) through PIP2. This interaction allows both Ca(2+)-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca(2+) triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca(2+) influx bringing the vesicle membrane close enough for membrane fusion.