Daniel Kümmel

Principles of membrane tethering and fusion in endosome and lysosome biogenesis.

Endosomes and lysosomes receive cargo via vesicular carriers that arrive along multiple trafficking routes. On both organelles, tethering proteins have been identified that interact specifically with Rab5 on endosomes and Rab7 on late endosomes/lysosomes and that facilitate the SNARE-driven membrane fusion. Even though the structure and stoichiometry of the involved proteins and protein complexes differ strongly, they may operate by similar principles. Within this review, we will provide insights into their common functions and discuss the open questions in the field.

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.

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis‐Golgi membrane. The crystal structure of human BET3 has been determined to 1.55‐Å resolution. BET3 adopts an α/β‐plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane‐associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein–protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.

Structure of D-63 from Sulfolobus Spindle-Shaped Virus 1: Surface Properties of the Dimeric Four-Helix Bundle Suggest an Adaptor Protein Function

ABSTRACT Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH ≤ 4.0) hot springs (≥70°C) around the world. SSV1 contains a 15.5-kb double-stranded DNA genome that encodes 34 proteins with greater than 50 amino acids. A site-specific integrase and a DnaA-like protein have been previously identified by sequence homology, and three structural proteins have been isolated from purified virus and identified by N-terminal sequencing (VP1, VP2, and VP3). The functions of the remaining 29 proteins are currently unknown. To assign functions to these proteins, we have initiated biochemical and structural studies on the SSV1 proteome. Here we report the structure of SSV1 D-63. The structure reveals a helix-turn-helix motif that dimerizes to form an antiparallel four-helix bundle. Mapping residues conserved among three fusellovirus isolates onto the structure shows that one face of the rod-shaped molecule is highly conserved. This conserved surface spans the dimer axis and thus exhibits 2-fold symmetry. Two smaller conserved patches, also related by 2-fold symmetry, are found on the opposite face of the molecule. All of these conserved surfaces are devoid of clefts or pockets typically used to bind small molecules, suggesting that D-63 may function as an adaptor protein in macromolecular assembly.

Unique self-palmitoylation activity of the transport protein particle component Bet3: A mechanism required for protein stability

Bet3 is a component of the transport protein particle complex involved in vesicular trafficking to and through the Golgi complex. X-ray structural analysis of human and mouse Bet3 revealed a hydrophobic tunnel inside the protein, which is occupied by a fatty acid linked to cysteine-68. We show here that Bet3 has strong self-palmitoylating activity. Incubation of purified Bet3 with [3H]palmitoyl-CoA (Pal-CoA) leads to a rapid and stoichiometric attachment of fatty acids to cysteine-68. Bet3 has an intrinsic affinity for Pal-CoA, and the palmitoylation reaction occurs at physiological pH values and Pal-CoA concentrations. Moreover, Bet3 is also efficiently palmitoylated at cysteine-68 inside vertebrate cells. Palmitoylation can occur late after Bet3 synthesis, but once the fatty acids are bound they are not removed, not even by disassembly of the Golgi complex. Narrowing the hydrophobic tunnel by exchange of alanine-82 with bulkier amino acids inhibits palmitoylation, both in vitro and inside cells, indicating that the fatty acid must insert into the tunnel for stable attachment. Finally, we show that palmitoylation of Bet3 plays a structural role. CD spectroscopy reveals that chemically deacylated Bet3 has a reduced melting temperature. As a consequence of its structural defect nonacylated Bet3 does not bind to TPC6, a further subunit of the transport protein particle complex, and is degraded inside cells.

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.

The Structure of the Trapp Subunit Tpc6 Suggests a Model for a Trapp Subcomplex.

The TRAPP (transport protein particle) complexes are tethering complexes that have an important role at the different steps of vesicle transport. Recently, the crystal structures of the TRAPP subunits SEDL and BET3 have been determined, and we present here the 1.7 Å crystal structure of human TPC6, a third TRAPP subunit. The protein adopts an α/β‐plait topology and forms a dimer. In spite of low sequence similarity, the structure of TPC6 strikingly resembles that of BET3. The similarity is especially prominent at the dimerization interfaces of the proteins. This suggests heterodimerization of TPC6 and BET3, which is shown by in vitro and in vivo association studies. Together with TPC5, another TRAPP subunit, TPC6 and BET3 are supposed to constitute a family of paralogous proteins with closely similar three‐dimensional structures but little sequence similarity among its members.

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

Significance Synaptic vesicle fusion at synapses is the primary mechanism by which neurons communicate. A highly conserved membrane fusion machine known as the SNARE complex mediates this process. In addition, neuronal SNARE-binding regulatory proteins have evolved to control the kinetics and speed of SNARE assembly at synapses. One such SNARE-binding protein, Complexin, has been found to inhibit synaptic vesicle fusion in the absence of an action potential and activate SNARE-mediated release during stimulation. Here we examine molecular models for how Complexin can mediate these unique effects on the SNARE fusion machine using genetic rescue experiments in Drosophila. We find that alternate SNARE-binding mechanisms by Complexin are likely to contribute to distinct inhibitory and activating functions in vivo. Complexin (Cpx) is a SNARE-binding protein that regulates neurotransmission by clamping spontaneous synaptic vesicle fusion in the absence of Ca2+ 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 Ca2+ 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.

Conformational dynamics of calcium-triggered activation of fusion by synaptotagmin.

Synaptotagmin triggers rapid exocytosis of neurotransmitters from synaptic vesicles in response to Calcium (Ca(2+)) ions. Here, we use a novel Nanodisc-based system, designed to be a soluble mimetic of the clamped synaptic vesicle-bilayer junction, combined with fluorescence resonance energy transfer (FRET) spectroscopy to monitor the structural relationships among SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), Synaptotagmin C2 domains, and the lipid bilayer in real time during the Ca(2+)-activation process. We report that Synaptotagmin remains rigidly fixed on the partially assembled SNARE complex with no detectable internal rearrangement of its C2 domains, even as it rapidly inserts into the bilayer. We hypothesize that this straightforward, one-step physical mechanism could explain how this Ca(2+)- sensor rapidly activates neurotransmitter release from the clamped state.

The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p

The exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins to confer both specificity and directionality to vesicular traffic.