TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation.

The TSC complex is a critical negative regulator of the small GTPase Rheb and mTORC1 in cellular stress signaling. The TSC2 subunit contains a catalytic GTPase activating protein domain and interacts with multiple regulators, while the precise function of TSC1 is unknown. Here we provide a structural characterization of TSC1 and define three domains: a C-terminal coiled-coil that interacts with TSC2, a central helical domain that mediates TSC1 oligomerization, and an N-terminal HEAT repeat domain that interacts with membrane phosphatidylinositol phosphates (PIPs). TSC1 architecture, oligomerization, and membrane binding are conserved in fungi and humans. We show that lysosomal recruitment of the TSC complex and subsequent inactivation of mTORC1 upon starvation depend on the marker lipid PI3,5P2, demonstrating a role for lysosomal PIPs in regulating TSC complex and mTORC1 activity via TSC1. Our study thus identifies a vital role of TSC1 in TSC complex function and mTORC1 signaling.

Structure of the metazoan Rab7 GEF complex Mon1-Ccz1-Bulli.

The endosomal system of eukaryotic cells represents a central sorting and recycling compartment linked to metabolic signaling and the regulation of cell growth. Tightly controlled activation of Rab GTPases is required to establish the different domains of endosomes and lysosomes. In metazoans, Rab7 controls endosomal maturation, autophagy, and lysosomal function. It is activated by the guanine nucleotide exchange factor (GEF) complex Mon1-Ccz1-Bulli (MCBulli) of the tri-longin domain (TLD) family. While the Mon1 and Ccz1 subunits have been shown to constitute the active site of the complex, the role of Bulli remains elusive. We here present the cryo-electron microscopy (cryo-EM) structure of MCBulli at 3.2 Å resolution. Bulli associates as a leg-like extension at the periphery of the Mon1 and Ccz1 heterodimers, consistent with earlier reports that Bulli does not impact the activity of the complex or the interactions with recruiter and substrate GTPases. While MCBulli shows structural homology to the related ciliogenesis and planar cell polarity effector (Fuzzy-Inturned-Wdpcp) complex, the interaction of the TLD core subunits Mon1-Ccz1 and Fuzzy-Inturned with Bulli and Wdpcp, respectively, is remarkably different. The variations in the overall architecture suggest divergent functions of the Bulli and Wdpcp subunits. Based on our structural analysis, Bulli likely serves as a recruitment platform for additional regulators of endolysosomal trafficking to sites of Rab7 activation.

Regulatory sites in the Mon1-Ccz1 complex control Rab5 to Rab7 transition and endosome maturation.

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here, we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in Drosophila nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, we further identify a conserved Rab5-binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system.

Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation.

Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate-binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.

Targeting of the Mon1-Ccz1 Rab guanine nucleotide exchange factor to distinct organelles by a synergistic protein and lipid code.

Activation of the small GTPase Rab7 by its cognate guanine nucleotide exchange factor Mon1-Ccz1 (MC1) is a key step in the maturation of endosomes and autophagosomes. This process is tightly regulated and subject to precise spatiotemporal control of MC1 localization, but the mechanisms that underly MC1 localization have not been fully elucidated. We here identify and characterize an amphipathic helix in Ccz1, which is required for the function of Mon-Ccz1 in autophagy, but not endosomal maturation. Furthermore, our data show that the interaction of the Ccz1 amphipathic helix with lipid packing defects, binding of Mon1 basic patches to positively charged lipids, and association of MC1 with recruiter proteins collectively govern membrane recruitment of the complex in a synergistic and redundant manner. Membrane binding enhances MC1 activity predominantly by increasing enzyme and substrate concentration on the membrane, but interaction with recruiter proteins can further stimulate the guanine nucleotide exchange factor. Our data demonstrate that specific protein and lipid cues convey the differential targeting of MC1 to endosomes and autophagosomes. In conclusion, we reveal the molecular basis for how MC1 is adapted to recognize distinct target compartments by exploiting the unique biophysical properties of organelle membranes and thus provide a model for how the complex is regulated and activated independently in different functional contexts.

Molecular insights into endolysosomal microcompartment formation and maintenance.

The endolysosomal system of eukaryotic cells has a key role in the homeostasis of the plasma membrane, in signaling and nutrient uptake, and is abused by viruses and pathogens for entry. Endocytosis of plasma membrane proteins results in vesicles, which fuse with the early endosome. If destined for lysosomal degradation, these proteins are packaged into intraluminal vesicles, converting an early endosome to a late endosome, which finally fuses with the lysosome. Each of these organelles has a unique membrane surface composition, which can form segmented membrane microcompartments by membrane contact sites or fission proteins. Furthermore, these organelles are in continuous exchange due to fission and fusion events. The underlying machinery, which maintains organelle identity along the pathway, is regulated by signaling processes. Here, we will focus on the Rab5 and Rab7 GTPases of early and late endosomes. As molecular switches, Rabs depend on activating guanine nucleotide exchange factors (GEFs). Over the last years, we characterized the Rab7 GEF, the Mon1-Ccz1 (MC1) complex, and key Rab7 effectors, the HOPS complex and retromer. Structural and functional analyses of these complexes lead to a molecular understanding of their function in the context of organelle biogenesis.

Structural analysis revealed a novel conformation of the NTRC reductase domain from Chlamydomonas reinhardtii.

In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates.

Visible-Light Removable Photocaging Groups Accepted by MjMAT Variant: Structural Basis and Compatibility with DNA and RNA Methyltransferases.

Methylation and demethylation of DNA, RNA and proteins constitutes a major regulatory mechanism in epigenetic processes. Investigations would benefit from the ability to install photo-cleavable groups at methyltransferase target sites that block interactions with reader proteins until removed by non-damaging light in the visible spectrum. Engineered methionine adenosyltransferases (MATs) have been exploited in cascade reactions with methyltransferases (MTases) to modify biomolecules with non-natural groups, including first evidence for accepting photo-cleavable groups. We show that an engineered MAT from Methanocaldococcus jannaschii (PC-MjMAT) is 308-fold more efficient at converting ortho-nitrobenzyl-(ONB)-homocysteine than the wildtype enzyme. PC-MjMAT is active over a broad range of temperatures and compatible with MTases from mesophilic organisms. We solved the crystal structures of wildtype and PC-MjMAT in complex with AdoONB and a red-shifted derivative thereof. These structures reveal that aromatic stacking interactions within the ligands are key to accommodating the photocaging groups in PC-MjMAT. The enlargement of the binding pocket eliminates steric clashes to enable AdoMet analogue binding. Importantly, PC-MjMAT exhibits remarkable activity on methionine analogues with red-shifted ONB-derivatives enabling photo-deprotection of modified DNA by visible light.

Structure of membrane tethers and their role in fusion.

Vesicular transport between different membrane compartments is a key process in cell biology required for the exchange of material and information. The complex machinery that executes the formation and delivery of transport vesicles has been intensively studied and yielded a comprehensive view of the molecular principles that underlie the budding and fusion process. Tethering also represents an essential step in each trafficking pathway. It is mediated by Rab GTPases in concert with so-called tethering factors, which constitute a structurally diverse family of proteins that share a similar role in promoting vesicular transport. By simultaneously binding to proteins and/or lipids on incoming vesicles and the target compartment, tethers are thought to bridge donor and acceptor membrane. They thus provide specificity while also promoting fusion. However, how tethering works at a mechanistic level is still elusive. We here discuss the recent advances in the structural and biochemical characterization of tethering complexes that provide novel insight on how these factors might contribute the efficiency of fusion.

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein.

Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein-splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of non-redundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class-3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high-resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.

Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with Photocaging Groups and Reversible DNA Modification in Cascade Reactions.

Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site-specific inhibition and timed removal. S-Adenosyl-L-methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC-ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC-ChMAT at 1.87 Šrevealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC-MATs were compatible with DNA- and RNA-MTases, enabling sequence-specific modification ("writing") of plasmid DNA and light-triggered removal ("erasing").

Comparison of the functional and structural characteristics of rare TSC2 variants with clinical and genetic findings.

The TSC1 and TSC2 gene products interact to form the tuberous sclerosis complex (TSC), an important negative regulator of the mechanistic target of rapamycin complex 1 (TORC1). Inactivating mutations in TSC1 or TSC2 cause TSC, and the identification of a pathogenic TSC1 or TSC2 variant helps establish a diagnosis of TSC. However, it is not always clear whether TSC1 and TSC2 variants are inactivating. To determine whether TSC1 and TSC2 variants of uncertain clinical significance affect TSC complex function and cause TSC, in vitro assays of TORC1 activity can be employed. Here we combine genetic, functional, and structural approaches to try and classify a series of 15 TSC2 VUS. We investigated the effects of the variants on the formation of the TSC complex, on TORC1 activity and on TSC2 pre-mRNA splicing. In 13 cases (87%), the functional data supported the hypothesis that the identified TSC2 variant caused TSC. Our results illustrate the benefits and limitations of functional testing for TSC.

A guanine nucleotide exchange factor (GEF) limits Rab GTPase-driven membrane fusion.

The identity of organelles in the endomembrane system of any eukaryotic cell critically depends on the correctly localized Rab GTPase, which binds effectors and thus promotes membrane remodeling or fusion. However, it is still unresolved which factors are required and therefore define the localization of the correct fusion machinery. Using SNARE-decorated proteoliposomes that cannot fuse on their own, we now demonstrate that full fusion activity can be achieved by just four soluble factors: a soluble SNARE (Vam7), a guanine nucleotide exchange factor (GEF, Mon1-Ccz1), a Rab-GDP dissociation inhibitor (GDI) complex (prenylated Ypt7-GDI), and a Rab effector complex (HOPS). Our findings reveal that the GEF Mon1-Ccz1 is necessary and sufficient for stabilizing prenylated Ypt7 on membranes. HOPS binding to Ypt7-GTP then drives SNARE-mediated fusion, which is fully GTP-dependent. We conclude that an entire fusion cascade can be controlled by a GEF.

Structure of the Tuberous Sclerosis Complex 2 (TSC2) N Terminus Provides Insight into Complex Assembly and Tuberous Sclerosis Pathogenesis.

Tuberous sclerosis complex (TSC) is caused by mutations in the TSC1 and TSC2 tumor suppressor genes. The gene products hamartin and tuberin form the TSC complex that acts as GTPase-activating protein for Rheb and negatively regulates the mammalian target of rapamycin complex 1 (mTORC1). Tuberin contains a RapGAP homology domain responsible for inactivation of Rheb, but functions of other protein domains remain elusive. Here we show that the TSC2 N terminus interacts with the TSC1 C terminus to mediate complex formation. The structure of the TSC2 N-terminal domain from Chaetomium thermophilum and a homology model of the human tuberin N terminus are presented. We characterize the molecular requirements for TSC1-TSC2 interactions and analyze pathological point mutations in tuberin. Many mutations are structural and produce improperly folded protein, explaining their effect in pathology, but we identify one point mutant that abrogates complex formation without affecting protein structure. We provide the first structural information on TSC2/tuberin with novel insight into the molecular function.

Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo.

Complexin (Cpx) is a SNARE-binding protein that regulates neurotransmission by clamping spontaneous synaptic vesicle fusion in the absence of Ca(2+) influx while promoting evoked release in response to an action potential. Previous studies indicated Cpx may cross-link multiple SNARE complexes via a trans interaction to function as a fusion clamp. During Ca(2+) influx, Cpx is predicted to undergo a conformational switch and collapse onto a single SNARE complex in a cis-binding mode to activate vesicle release. To test this model in vivo, we performed structure-function studies of the Cpx protein in Drosophila. Using genetic rescue approaches with cpx mutants that disrupt SNARE cross-linking, we find that manipulations that are predicted to block formation of the trans SNARE array disrupt the clamping function of Cpx. Unexpectedly, these same mutants rescue action potential-triggered release, indicating trans-SNARE cross-linking by Cpx is not a prerequisite for triggering evoked fusion. In contrast, mutations that impair Cpx-mediated cis-SNARE interactions that are necessary for transition from an open to closed conformation fail to rescue evoked release defects in cpx mutants, although they clamp spontaneous release normally. Our in vivo genetic manipulations support several predictions made by the Cpx cross-linking model, but unexpected results suggest additional mechanisms are likely to exist that regulate Cpx's effects on SNARE-mediated fusion. Our findings also indicate that the inhibitory and activating functions of Cpx are genetically separable, and can be mapped to distinct molecular mechanisms that differentially regulate the SNARE fusion machinery.

The Habc domain of the SNARE Vam3 interacts with the HOPS tethering complex to facilitate vacuole fusion.

Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the Habc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 Habc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the Habc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the Habc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the Habc domain. In agreement, membranes carrying Vam3ΔHabc fuse poorly unless an excess of HOPS is provided. These data suggest that the Habc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion.

Structural identification of the Vps18 ß-propeller reveals a critical role in the HOPS complex stability and function.

Membrane fusion at the vacuole, the lysosome equivalent in yeast, requires the HOPS tethering complex, which is recruited by the Rab7 GTPase Ypt7. HOPS provides a template for the assembly of SNAREs and thus likely confers fusion at a distinct position on vacuoles. Five of the six subunits in HOPS have a similar domain prediction with strong similarity to COPII subunits and nuclear porins. Here, we show that Vps18 indeed has a seven-bladed ß-propeller as its N-terminal domain by revealing its structure at 2.14 Å. The Vps18 N-terminal domain can interact with the N-terminal part of Vps11 and also binds to lipids. Although deletion of the Vps18 N-terminal domain does not preclude HOPS assembly, as revealed by negative stain electron microscopy, the complex is instable and cannot support membrane fusion in vitro. We thus conclude that the ß-propeller of Vps18 is required for HOPS stability and function and that it can serve as a starting point for further structural analyses of the HOPS tethering complex.

A half-zippered SNARE complex represents a functional intermediate in membrane fusion.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate fusion by pulling biological membranes together via a zippering mechanism. Recent biophysical studies have shown that t- and v-SNAREs can assemble in multiple stages from the N-termini toward the C-termini. Here we show that functionally, membrane fusion requires a sequential, two-step folding pathway and assign specific and distinct functions for each step. First, the N-terminal domain (NTD) of the v-SNARE docks to the t-SNARE, which leads to a conformational rearrangement into an activated half-zippered SNARE complex. This partially assembled SNARE complex locks the C-terminal (CTD) portion of the t-SNARE into the same structure as in the postfusion 4-helix bundle, thereby creating the binding site for the CTD of the v-SNARE and enabling fusion. Then zippering of the remaining CTD, the membrane-proximal linker (LD), and transmembrane (TMD) domains is required and sufficient to trigger fusion. This intrinsic property of the SNAREs fits well with the action of physiologically vital regulators such as complexin. We also report that NTD assembly is the rate-limiting step. Our findings provide a refined framework for delineating the molecular mechanism of SNARE-mediated membrane fusion and action of regulatory proteins.

Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate.

Rab GTPases are key regulators of membrane traffic pathways within eukaryotic cells. They are specifically activated by guanine nucleotide exchange factors (GEFs), which convert them from their "inactive" GDP-bound form to the "active" GTP-bound form. In higher eukaryotes, proteins containing DENN-domains comprise a major GEF family. Here we describe at 2.1-Å resolution the first structure of a DENN-domain protein, DENND1B-S, complexed with its substrate Rab35, providing novel insights as to how DENN-domain GEFs interact with and activate Rabs. DENND1B-S is bi-lobed, and interactions with Rab35 are through conserved surfaces in both lobes. Rab35 binds via switch regions I and II, around the nucleotide-binding pocket. Positional shifts in Rab residues required for nucleotide binding may lower its affinity for bound GDP, and a conformational change in switch I, which makes the nucleotide-binding pocket more solvent accessible, likely also facilitates exchange.

κB-Ras proteins are fast-exchanging GTPases and function via nucleotide-independent binding of Ral GTPase-activating protein complexes.

κB-Ras (NF-κB inhibitor-interacting Ras-like protein) GTPases are small Ras-like GTPases but harbor interesting differences in important sequence motifs. They act in a tumor-suppressive manner as negative regulators of Ral (Ras-like) GTPase and NF-κB signaling, but little is known about their mode of function. Here, we demonstrate that, in contrast to predictions based on primary structure, κB-Ras GTPases possess hydrolytic activity. Combined with low nucleotide affinity, this renders them fast-cycling GTPases that are predominantly GTP-bound in cells. We characterize the impact of κB-Ras mutations occurring in tumors and demonstrate that nucleotide binding affects κB-Ras stability but is not strictly required for RalGAP (Ral GTPase-activating protein) binding. This demonstrates that κB-Ras control of RalGAP/Ral signaling occurs in a nucleotide-binding- and switch-independent fashion.