Biology subjects

Raunser, S.

Publications and source records attributed to Raunser, S..

45 records · Page 3Linked to original sources

bioRxiv2019

Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

Actin undergoes structural transitions during polymerization, ATP hydrolysis and subsequent release of inorganic phosphate. Several actin binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin binding proteins. Furthermore, high-resolution cryo-EM structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/794495v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (47K):\norg.highwire.dtl.DTLVardef@1a81b98org.highwire.dtl.DTLVardef@1aba96borg.highwire.dtl.DTLVardef@ffc218org.highwire.dtl.DTLVardef@11fe454_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFive high-resolution cryo-EM structures of stabilized filamentous actin\nC_LIO_LIPhalloidin traps different structural states depending on when it is added\nC_LIO_LIThe effect of phalloidin and jasplakinolide on filamentous actin is not identical\nC_LIO_LIBoth toxins likely interfere with the binding of proteins sensing F-actins nucleotide state\nC_LI

biochemistry↗

bioRxiv2019

Profilin and formin constitute a pacemaker system for robust actin filament growth

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

biochemistry↗

bioRxiv2019

Towards the application of Tc toxins as a universal protein translocation system

Tc toxins are large bacterial protein complexes that inject cytotoxic enzymes into target cells using a sophisticated syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigated whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely human Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, human Ras-binding domain of CRAF kinase, and tobacco etch virus protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to successfully take advantage of this new universal protein translocation system.

biochemistry↗

bioRxiv2019

Cryo-EM structure of the ClpXP protein degradation machinery

The ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and eukaryotes. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the first high-resolution cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXPs complex mode of action and provides a structural framework for future pharmacological applications.

molecular biology↗

bioRxiv2019

Conserved architecture of Tc toxins from human and insect pathogenic bacteria

Tc toxin complexes use a syringe-like mechanism to penetrate the membrane and translocate a toxic enzyme into the host cytosol. They are composed of three components: TcA, TcB and TcC. Until recently, low-resolution structures of TcA from different bacteria suggested that Tc toxins differ considerably in their architecture and possibly in their mechanism of action. Here, we present high-resolution structures and functional studies of five TcAs from different insect and human pathogenic bacteria. Contrary to previous expectations, their overall composition and domain organization is almost identical. The TcAs assemble as a pentamer with a central -helical channel surrounded by a shell composed of conserved -helical domains and variable {beta}-sheet domains. Essential structural features, including a conserved trefoil protein knot, are present in all five TcAs, suggesting a common mechanism of action. All TcAs form functional pores and can be combined with TcB-TcC subunits from other species resulting in chimeric holotoxins. We have identified a conserved ionic pair that stabilizes the shell, likely operating as a strong latch that only springs open after the destabilization of other regions. Our results lead to new insights into the architecture and host specificity of the Tc toxin family.

molecular biology↗

bioRxiv2019

Structure of a Tc holotoxin pore provides insights into the translocation mechanism

Tc toxins are modular toxin systems that are composed of a pentameric membrane translocator (TcA) and a cocoon (TcB and TcC) encapsulating the toxic enzyme. Binding of Tcs to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. Here, we show in atomic detail an assembled Tc toxin complex from P. luminescens in the membrane. We find that the five TcA protomers conformationally adapt to fit around the cocoon during prepore-to-pore transition. The architecture of the Tc toxin complex also allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Mammalian lipids with zwitterionic head groups are preferred over other lipids for Tc toxin integration. The translocated toxic enzyme, which can be partially visualized, transiently interacts with alternating negative charges and hydrophobic stretches.

molecular biology↗

bioRxiv2018

SPHIRE-crYOLO: A fast and well-centering automated particle picker for cryo-EM

Selecting particles from digital micrographs is an essential step in single particle electron cryomicroscopy (cryo-EM). Since manual selection is a tedious and time-consuming process, many automatic particle pickers have been developed. However, they have problems especially with non-ideal datasets. Here, we present a novel automated particle picking software called crYOLO, which is based on the deep learning object detection system \"You Only Look Once\" (YOLO). After training the network with 500 - 2,500 particles per dataset, it automatically selects the particles with high accuracy and precision reaching a speed of up to six micrographs per second. Importantly, we show that crYOLO can be trained to select previously unseen datasets paving the way for completely automated \"on-the-fly\" cryo-EM data pre-processing during data acquisition. CrYOLO is available as standalone program under http://sphire.mpg.de/ and will be part of the image processing workflow in SPHIRE.

biophysics↗

bioRxiv2018

Membrane insertion of α-xenorhabdolysin in near-atomic detail

-Xenorhabdolysins (Xax) are -pore-forming toxins (-PFT) from pathogenic bacteria that form 1-1.3 MDa large pore complexes to perforate the host cell membrane. PFTs are used by a variety of bacterial pathogens as an offensive or defensive mechanism to attack host cells. Due to the lack of structural information, the molecular mechanism of action of Xax toxins is poorly understood. Here, we report the cryo-EM structure of the XaxAB pore complex from Xenorhabdus nematophila at an average resolution of 4.0 [A] and the crystal structures of the soluble monomers of XaxA and XaxB at 2.5 [A] and 3.4 [A], respectively. The structures reveal that XaxA and XaxB are built similarly and appear as heterodimers in the 12-15 subunits containing pore. The structure of the XaxAB pore represents therefore the first structure of a bi-component -PFT. Major conformational changes in XaxB, including the swinging out of an amphipathic helix are responsible for membrane insertion. XaxA acts as an activator and stabilizer for XaxB that forms the actual transmembrane pore. Based on our results, we propose a novel structural model for the mechanism of action of Xax toxins.

molecular biology↗

bioRxiv2018

Electron cryo-microscopy structure of the canonical TRPC4 ion channel

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 [A]. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

biochemistry↗