Structural and biochemical studies of class C Vps tethering complexes HOPS and CORVET

In eukaryotischen Zellen werden einzelne Kompartimente mittels vesikulären Transportes verknüpft. Tethering dient dabei als Bindeglied zwischen Transport /Adres-sierung und Fusion mit der Zielmembran. Die Class C Vps Tethering- Komplexe HOPS und CORVET erfüllen diese Aufgabe im endolysosomalen Transportweg. Auch wenn die Funktion der Komplexe gut untersucht ist, so ist unser Wissen um die molekularen Grundlagen der Funktionsweise dieser Komplexe noch lückenhaft. Struktur-Funktions-Analysen können einen Einblick in diese Grundlagen gewähren. Für fünf der sechs Untereinheiten von HOPS wurden zueinander stark ähnliche Domänenarchitekturen vorhergesagt, was jüngst für den C-terminalen Bereich belegt werden konnte. In der vorliegenden Arbeit konnte nun erstmalig durch Kristallisation des N-terminus von Vps18 bestätigt werden, dass die Vorhersage der N-terminalen Domäne als β-Propeller korrekt ist. Das im Kristall als Dimer vorliegende Protein zeigt einen typischen siebenblättrigen β-Propeller mit einem Velcro-Verschluss. Die einzel¬nen Moleküle sind dabei zueinander um 180° gedreht und durch eine C2-Symmetrie verknüpft. Die Kontaktfläche des Dimers ist durch elektrostatische Interaktionen geprägt, jedoch ist die physiologische Relevanz der Interaktionsfläche und der beteiligten Aminosäuren trotz zum Teil hoher Konservierung fragwürdig. Vorhersagen und experimentellen Daten nach handelt es sich aller Wahrscheinlichkeit nach um Kristall-kontakte. Am Rand des Propellers, genauer zwischen Blatt 2 und Blatt 3, konnte eine auffällige Schleifenstruktur identifiziert werden, welche eine zwischen Spezies konser- vier¬te Sequenz Lx[KR][WFLI]K (in Hefe LNKIK) aufweist. Durch Lipid-Co- Sedimentationsexperimente sowie gezielte Mutagenese konnte gezeigt werden, dass die beiden Lysine dieser Sequenz entscheidend für die Membranbindung sind. Somit konnte in dieser Arbeit erstmalig dem N-terminalen Bereich eine Funktion außerhalb des Komplexaufbaus zugewiesen werden, sowie eine neue Klasse von Membran-bindenden β-Propellern, neben den bekannten PROPPINs, identifiziert werden. Die bekannte Beteiligung an der Stabilisierung des HOPS- Komplexes konnte im Rahmen von in vitro-Experimenten genauer eingegrenzt werden. Diese zeigten eine direkte Interaktion des N-terminalen Propellers von Vps18 mit der N-terminalen Domäne von Vps11. Die N-terminale Domäne von Vps11 weist zwar weder eine Lx[KR][WFLI]K-Sequenz noch ein PROPPIN-Motiv auf, dennoch konnte eine Membranaffinität festgestellt werden. Aufgrund des Fehlens einer Kristallstruktur konnte der interagierende Bereich jedoch nicht durch gezielte Mutagenese-Studien ermittelt werden. Der genaue Bindungsmechanismus bleibt somit für Vps11, im Gegensatz zu Vps18, im Unklaren.In eukaryotic cells, individual compartments are connected via the vesicular transport pathway. Tethering acts as the bridging step between transport/addressing of vesicles and their fusion with target membranes. The class C Vps tethering complexes HOPS and CORVET are acting on the endolysomal transport pathway. Although their cellular function is well understood, little is known about the molecular mechanisms. Structure-function analysis can be of great help to further our molecular understanding of these tethering mechanisms. Five of the six known subunits of HOPS share similar predicted domain architectures. At least for the C-terminal domain, this prediction was recently confirmed by protein crystallography. In the present study, the N-terminal domain of the HOPS subunit Vps18 was crystallized. The resulting structure is that of a typical seven-bladed β-propeller, with a velcro closure, thus confirming the initial structure prediction. In the asymmetric unit of the crystal, a dimer of two molecules is observed, related to each other by a 180° rotation and C2 symmetry. The interface is defined by electrostatic interactions. Although several of the interacting residues show high levels of conserva-tion, experimental data and predictions indicate that the interaction is not likely to be of relevance for the biological function of Vps18. At the rim of the propeller, more specifically between blade 2 and blade 3, an extended loop containing a conserved Lx[KR][WFLI]K (in yeast LNKIK) sequence can be found. Relying on lipid co-sedimentation assays and mutagenesis, it could be shown that this sequence is responsible for interactions of the propeller with lipid membranes. Thus for the first time, a functional role of the N-terminal domain could be determined, apart from its known function in stabilizing the complex. Moreover, the Vps18 β-propeller constitutes a novel class of membrane-binding propellers, complementing the known PROPPINs. Besides this, the known function of stabilizing the HOPS complex could be further specified by showing that the Vps18 propeller can interact directly with the N-terminal domain of Vps11. Although Vps11 shows neither a Lx[KR][WFLI]K sequence nor a PROPPIN motif, membrane-binding properties were also found for this N terminal domain. Due to the lack of a known structure, no further analysis of this property was possible. Therefore, and different from the Vps18 propeller, the lipid-binding mechanism of Vps11 remains unknown

The Optimized Link State Routing Protocol Evaluation through Experiments and Simulation

In this paper, we describe the Optimized Link State Routing Protocol (OLSR) [1] for Mobile Ad-hoc NETworks (MANETs) and the evaluation of this protocol through experiments and simulations. In particular, we emphasize the practical tests and intensive simulations, which have been used in guiding and evaluating the design of the protocol, and which have been a key to identifying both problems and solutions.\ud \ud OLSR is a proactive link-state routing protocol, employing periodic message exchange for updating topological information in each node in the network. I.e. topological information is flooded to all nodes in the network.\ud \ud Conceptually, OLSR contains three elements: Mechanisms\ud for neighbor sensing based on periodic exchange of HELLO\ud messages within a node’s neighborhood. Generic mechanisms\ud for efficient flooding of control traffic into the network employing the concept of multipoint relays (MPRs) [5] for a significant reduction of duplicate retransmissions during the flooding process. And a specification of a set of control-messages providing each node with sufficient topological information to be able to compute an optimal route to each destination in the network using any shortest-path algorithm.\ud Experimental work, running a test-network of laptops with IEEE 802.11 wireless cards, revealed interesting properties. While the protocol, as originally specified, works quite well, it was found, that enforcing “jitter” on the interval between the periodic exchange of control messages in OLSR and piggybacking said control messages into a single packet, significantly reduced the number of messages lost due to collisions. It was also observed, that under certain conditions a “naive” neighbor sensing mechanism was insufficient: a bad link between two nodes\ud (e.g. when two nodes are on the edge of radio range) might on occasion transmit a HELLO message in both directions (hence enabling the link for routing), while not being able to sustain continuous traffic. This would result in “route-flapping” and temporary loss of connectivity.\ud With the experimental results as basis, we have been deploying simulations to reveal the impact of the various algorithmic improvements, described above.\u

Directionality of PYD filament growth determined by the transition of NLRP3 nucleation seeds to ASC elongation

Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming nucleotide-binding oligomerization domain (NOD)-like receptors with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) mediate oligomerization into filamentous assemblies. We describe the cryo-electron microscopy (cryo-EM) structure of the human NLRP3(PYD) filament and identify a pattern of highly polar interface residues that form the homomeric interactions leading to characteristic filament ends designated as A- and B-ends. Coupling a titration polymerization assay to cryo-EM, we demonstrate that ASC adaptor protein elongation on NLRP3(PYD) nucleation seeds is unidirectional, associating exclusively to the B-end of the filament. Notably, NLRP3 and ASC PYD filaments exhibit the same symmetry in rotation and axial rise per subunit, allowing a continuous transition between NLRP3 and ASC. Integrating the directionality of filament growth, we present a molecular model of the ASC speck consisting of active NLRP3, ASC, and Caspase-1 proteins

A marine cryptochrome with an inverse photo-oligomerization mechanism

Abstract Cryptochromes (CRYs) are a structurally conserved but functionally diverse family of proteins that can confer unique sensory properties to organisms. In the marine bristle worm Platynereis dumerilii, its light receptive cryptochrome L-CRY (PdLCry) allows the animal to discriminate between sunlight and moonlight, an important requirement for synchronizing its lunar cycle-dependent mass spawning. Using cryo-electron microscopy, we show that in the dark, PdLCry adopts a dimer arrangement observed neither in plant nor insect CRYs. Intense illumination disassembles the dimer into monomers. Structural and functional data suggest a mechanistic coupling between the light-sensing flavin adenine dinucleotide chromophore, the dimer interface, and the C-terminal tail helix, with a likely involvement of the phosphate binding loop. Taken together, our work establishes PdLCry as a CRY protein with inverse photo-oligomerization with respect to plant CRYs, and provides molecular insights into how this protein might help discriminating the different light intensities associated with sunlight and moonlight

Direct Interaction of Avian Cryptochrome 4 with a Cone Specific G-Protein

Background: Night-migratory birds sense the Earth's magnetic field by an unknown molecular mechanism. Theoretical and experimental evidence support the hypothesis that the light-induced formation of a radical-pair in European robin cryptochrome 4a (ErCry4a) is the primary signaling step in the retina of the bird. In the present work, we investigated a possible route of cryptochrome signaling involving the alpha-subunit of the cone-secific heterotrimeric G protein from European robin. Methods: Protein-protein interaction studies include surface plasmon resonance, pulldown affinity binding and Forster resonance energy transfer. Results: Surface plasmon resonance studies showed direct interaction, revealing high to moderate affinity for binding of non-myristoylated and myristoylated G protein to ErCry4a, respectively. Pulldown affinity experiments confirmed this complex formation in solution. We validated these in vitro data by monitoring the interaction between ErCry4a and G protein in a transiently transfected neuroretinal cell line using Forster resonance energy transfer. Conclusions: Our results suggest that ErCry4a and the G protein also interact in living cells and might constitute the first biochemical signaling step in radical-pair-based magnetoreception