A marine cryptochrome with an inverse photo-oligomerization mechanism.

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 α-subunit of the cone-secific heterotrimeric G protein from European robin. METHODS: Protein-protein interaction studies include surface plasmon resonance, pulldown affinity binding and Förster 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 Förster 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.

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 NLRP3PYD 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 NLRP3PYD 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.

Structure of a collagen VI α3 chain VWA domain array: adaptability and functional implications of myopathy causing mutations.

Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein-protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales.

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

It takes two transducins to activate the cGMP-phosphodiesterase 6 in retinal rods.

Among cyclic nucleotide phosphodiesterases (PDEs), PDE6 is unique in serving as an effector enzyme in G protein-coupled signal transduction. In retinal rods and cones, PDE6 is membrane-bound and activated to hydrolyse its substrate, cGMP, by binding of two active G protein α-subunits (Gα*). To investigate the activation mechanism of mammalian rod PDE6, we have collected functional and structural data, and analysed them by reaction-diffusion simulations. Gα* titration of membrane-bound PDE6 reveals a strong functional asymmetry of the enzyme with respect to the affinity of Gα* for its two binding sites on membrane-bound PDE6 and the enzymatic activity of the intermediary 1 : 1 Gα* · PDE6 complex. Employing cGMP and its 8-bromo analogue as substrates, we find that Gα* · PDE6 forms with high affinity but has virtually no cGMP hydrolytic activity. To fully activate PDE6, it takes a second copy of Gα* which binds with lower affinity, forming Gα* · PDE6 · Gα*. Reaction-diffusion simulations show that the functional asymmetry of membrane-bound PDE6 constitutes a coincidence switch and explains the lack of G protein-related noise in visual signal transduction. The high local concentration of Gα* generated by a light-activated rhodopsin molecule efficiently activates PDE6, whereas the low density of spontaneously activated Gα* fails to activate the effector enzyme.

Mechanistic insights into the role of prenyl-binding protein PrBP/δ in membrane dissociation of phosphodiesterase 6.

Isoprenylated proteins are associated with membranes and their inter-compartmental distribution is regulated by solubilization factors, which incorporate lipid moieties in hydrophobic cavities and thereby facilitate free diffusion during trafficking. Here we report the crystal structure of a solubilization factor, the prenyl-binding protein (PrBP/δ), at 1.81 Å resolution in its ligand-free apo-form. Apo-PrBP/δ harbors a preshaped, deep hydrophobic cavity, capacitating apo-PrBP/δ to readily bind its prenylated cargo. To investigate the molecular mechanism of cargo solubilization we analyzed the PrBP/δ-induced membrane dissociation of rod photoreceptor phosphodiesterase (PDE6). The results suggest that PrBP/δ exclusively interacts with the soluble fraction of PDE6. Depletion of soluble species in turn leads to dissociation of membrane-bound PDE6, as both are in equilibrium. This "solubilization by depletion" mechanism of PrBP/δ differs from the extraction of prenylated proteins by the similar folded solubilization factor RhoGDI, which interacts with membrane bound cargo via an N-terminal structural element lacking in PrBP/δ.

Structural basis for λN-dependent processive transcription antitermination.

λN-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein λN, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a λN-NusA-NusB-NusE-nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and λN, validated by crosslinking/mass spectrometry. Due to intrinsic disorder, λN can act as a multiprotein/RNA interaction hub, which, together with nut site RNA, arranges NusA, NusB and NusE into a triangular complex. This complex docks via the NusA N-terminal domain and the λN C-terminus next to the RNA exit channel on RNA polymerase. Based on the structures, comparative crosslinking analyses and structure-guided mutagenesis, we hypothesize that λN mounts a multipronged strategy to reprogram the transcriptional machinery, which may include (1) the λN C terminus clamping the RNA exit channel, thus stabilizing the DNA:RNA hybrid; (2) repositioning of NusA and RNAP elements, thus redirecting nascent RNA and sequestering the upstream branch of a terminator hairpin; and (3) hindering RNA engagement of termination factor ρ and/or obstructing ρ translocation on the transcript.

Studies on the X-Ray and Solution Structure of FeoB from Escherichia coli BL21.

The ferrous iron transporter FeoB is an important factor in the iron metabolism of many bacteria. Although several structural studies have been performed on its cytosolic GTPase domain (NFeoB), the full-length structure of FeoB remains elusive. Based on a crystal packing analysis that was performed on crystals of NFeoB, a trimeric structure of the FeoB channel was proposed, where the transport pore runs along the trimer axis. Because this trimer has not been observed in some subsequently solved structures of NFeoB homologs, it remains unclear whether or not the trimer is indeed functionally relevant. Here, pulsed electron-electron double resonance spectroscopy, negative stain electron microscopy, and native mass spectrometry are used to analyze the oligomeric state of different soluble and full-length FeoB constructs. The results show that the full-length protein is predominantly monomeric, whereas dimers and trimers are formed to a small percentage. Furthermore, the solution structure of the switch I region is analyzed by pulsed electron-electron double resonance spectroscopy and a new, to our knowledge, crystal structure of NFeoB from Escherichia coli BL21 is presented.

Using Molecular Simulation to Model High-Resolution Cryo-EM Reconstructions.

An explosion of new data from high-resolution cryo-electron microscopy (cryo-EM) studies has produced a large number of data sets for many species of ribosomes in various functional states over the past few years. While many methods exist to produce structural models for lower resolution cryo-EM reconstructions, high-resolution reconstructions are often modeled using crystallographic techniques and extensive manual intervention. Here, we present an automated fitting technique for high-resolution cryo-EM data sets that produces all-atom models highly consistent with the EM density. Using a molecular dynamics approach, atomic positions are optimized with a potential that includes the cross-correlation coefficient between the structural model and the cryo-EM electron density, as well as a biasing potential preserving the stereochemistry and secondary structure of the biomolecule. Specifically, we use a hybrid structure-based/ab initio molecular dynamics potential to extend molecular dynamics fitting. In addition, we find that simulated annealing integration, as opposed to straightforward molecular dynamics integration, significantly improves performance. We obtain atomistic models of the human ribosome consistent with high-resolution cryo-EM reconstructions of the human ribosome. Automated methods such as these have the potential to produce atomistic models for a large number of ribosome complexes simultaneously that can be subsequently refined manually.

Structural snapshots of actively translating human ribosomes.

Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

Structure of the mammalian 80S initiation complex with initiation factor 5B on HCV-IRES RNA.

The universally conserved eukaryotic initiation factor (eIF) 5B, a translational GTPase, is essential for canonical translation initiation. It is also required for initiation facilitated by the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) RNA. eIF5B promotes joining of 60S ribosomal subunits to 40S ribosomal subunits bound by initiator tRNA (Met-tRNAi(Met)). However, the exact molecular mechanism by which eIF5B acts has not been established. Here we present cryo-EM reconstructions of the mammalian 80S-HCV-IRES-Met-tRNAi(Met)-eIF5B-GMPPNP complex. We obtained two substates distinguished by the rotational state of the ribosomal subunits and the configuration of initiator tRNA in the peptidyl (P) site. Accordingly, a combination of conformational changes in the 80S ribosome and in initiator tRNA facilitates binding of the Met-tRNAi(Met) to the 60S P site and redefines the role of eIF5B as a tRNA-reorientation factor.

Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement.

The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1A⋅Val-tRNA⋅GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection.

Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD2.

The dynamin-related Eps15-homology domain-containing protein 2 (EHD2) is a membrane-remodeling ATPase that regulates the dynamics of caveolae. Here, we established an electron paramagnetic resonance (EPR) approach to characterize structural features of membrane-bound EHD2. We show that residues at the tip of the helical domain can insert into the membrane and may create membrane curvature by a wedging mechanism. Using EPR and X-ray crystallography, we found that the N terminus is folded into a hydrophobic pocket of the GTPase domain in solution and can be released into the membrane. Cryoelectron microscopy demonstrated that the N terminus is not essential for oligomerization of EHD2 into a membrane-anchored scaffold. Instead, we found a function of the N terminus in regulating targeting and stable association of EHD2 to caveolae. Our data uncover an unexpected, membrane-induced regulatory switch in EHD2 and demonstrate the versatility of EPR to study structure and function of dynamin superfamily proteins.

Functional characterization of human myosin-18A and its interaction with F-actin and GOLPH3.

Molecular motors of the myosin superfamily share a generic motor domain region. They commonly bind actin in an ATP-sensitive manner, exhibit actin-activated ATPase activity, and generate force and movement in this interaction. Class-18 myosins form heavy chain dimers and contain protein interaction domains located at their unique N-terminal extension. Here, we characterized human myosin-18A molecular function in the interaction with nucleotides, F-actin, and its putative binding partner, the Golgi-associated phosphoprotein GOLPH3. We show that myosin-18A comprises two actin binding sites. One is located in the KE-rich region at the start of the N-terminal extension and appears to mediate ATP-independent binding to F-actin. The second actin-binding site resides in the generic motor domain and is regulated by nucleotide binding in the absence of intrinsic ATP hydrolysis competence. This core motor domain displays its highest actin affinity in the ADP state. Electron micrographs of myosin-18A motor domain-decorated F-actin filaments show a periodic binding pattern independent of the nucleotide state. We show that the PDZ module mediates direct binding of myosin-18A to GOLPH3, and this interaction in turn modulates the actin binding properties of the N-terminal extension. Thus, myosin-18A can act as an actin cross-linker with multiple regulatory modulators that targets interacting proteins or complexes to the actin-based cytoskeleton.

FHOD1 is a combined actin filament capping and bundling factor that selectively associates with actin arcs and stress fibers.

Formins are actin polymerization factors that are known to nucleate and elongate actin filaments at the barbed end. In the present study we show that human FHOD1 lacks actin nucleation and elongation capacity, but acts as an actin bundling factor with capping activity toward the filament barbed end. Constitutively active FHOD1 associates with actin filaments in filopodia and lamellipodia at the leading edge, where it moves with the actin retrograde flow. At the base of lamellipodia, FHOD1 is enriched in nascent, bundled actin arcs as well as in more mature stress fibers. This function requires actin-binding domains located N-terminally to the canonical FH1-FH2 element. The bundling phenotype is maintained in the presence of tropomyosin, confirmed by electron microscopy showing assembly of 5 to 10 actin filaments into parallel, closely spaced filament bundles. Taken together, our data suggest a model in which FHOD1 stabilizes actin filaments by protecting barbed ends from depolymerization with its dimeric FH2 domain, whereas the region N-terminal to the FH1 domain mediates F-actin bundling by simultaneously binding to the sides of adjacent F-actin filaments.

Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes.

Rasal, belonging to the GAP1 subfamily of Ras GTPase-activating proteins (RasGAPs) with dual RasGAP/RapGAP specificity, is epigenetically silenced in several tumor types. Surprisingly, the isolated protein has GAP activity on Rap but not on Ras. Its membrane recruitment is regulated by interaction with calcium and lipids, which simultaneously induces its RasGAP activity through a yet unknown mechanism. Here we show that the interaction of Rasal with membranes induces Rasal RasGAP activity by spatial and conformational regulation, although it does not have any effect on its RapGAP activity. Not only is colocalization of Rasal and Ras in the membrane essential for RasGAP activation, but direct and Ca-dependent interaction between the tandem C2 domains of Rasal and lipids of the membrane is also required. Whereas the C2A domain binds specifically phosphatidylserine, the C2B domain interacts with several phosphoinositol lipids. Finally we show, that similar to the C2 domains of synaptotagmins, the Rasal tandem C2 domains are able to sense and induce membrane curvature by the insertion of hydrophobic loops into the membrane.

Structure of the rigor actin-tropomyosin-myosin complex.

Regulation of myosin and filamentous actin interaction by tropomyosin is a central feature of contractile events in muscle and nonmuscle cells. However, little is known about molecular interactions within the complex and the trajectory of tropomyosin movement between its "open" and "closed" positions on the actin filament. Here, we report the 8 Å resolution structure of the rigor (nucleotide-free) actin-tropomyosin-myosin complex determined by cryo-electron microscopy. The pseudoatomic model of the complex, obtained from fitting crystal structures into the map, defines the large interface involving two adjacent actin monomers and one tropomyosin pseudorepeat per myosin contact. Severe forms of hereditary myopathies are linked to mutations that critically perturb this interface. Myosin binding results in a 23 Å shift of tropomyosin along actin. Complex domain motions occur in myosin, but not in actin. Based on our results, we propose a structural model for the tropomyosin-dependent modulation of myosin binding to actin.

Membrane fusion intermediates via directional and full assembly of the SNARE complex.

Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction, we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.

Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy.

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac ß-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.