Teneurins are ancient cell–cell adhesion receptors that are vital for brain development and synapse organisation. They originated in early metazoan evolution through a horizontal gene transfer event when a bacterial YD-repeat toxin fused to a eukaryotic receptor. We present X-ray crystallography and cryo-EM structures of two Teneurins, revealing a ~200 kDa extracellular super-fold in which eight sub-domains form an intricate structure centred on a spiralling YD-repeat shell. An alternatively spliced loop, which is implicated in homophilic Teneurin interaction and specificity, is exposed and thus poised for interaction. The N-terminal side of the shell is ‘plugged’ via a fibronectin-plug domain combination, which defines a new class of YD proteins. Unexpectedly, we find that these proteins are widespread amongst modern bacteria, suggesting early metazoan receptor evolution from a distinct class of proteins, which today includes both bacterial proteins and eukaryotic Teneurins.

Photosystem II (PSII) is a light-driven protein, involved in the primary reactions of photosynthesis. In plant photosynthetic membranes PSII forms large multisubunit supercomplexes, containing a dimeric core and up to four light-harvesting complexes (LHCs), which act as antenna proteins. Here we solved a three-dimensional (3D) structure of the CSM supercomplex from Arabidopsis thaliana using cryo-transmission electron microscopy (cryo-EM) and single-particle analysis at an overall resolution of 5.3 Å. Using a combination of homology modelling and restrained refinement against the cryo-EM map, it was possible to model atomic structures for all antenna complexes and almost all core subunits. We located all 35 chlorophylls of the core region based on the cyanobacterial PSII structure, whose positioning is highly conserved, as well as all the chlorophylls of the LHCII S and M trimers. A total of 13 and 9 chlorophylls were identified in CP26 and CP24, respectively. Energy flow from LHC complexes to the PSII reaction centre is proposed to follow preferential pathways: CP26 and CP29 directly transfer to the core using several routes for efficient transfer; the S trimer is directly connected to CP43 and the M trimer can efficiently transfer energy to the core through CP29 and the S trimer.

Enzymes of the six-transmembrane epithelial antigen of the prostate (STEAP) family reduce Fe3+ and Cu2+ ions to facilitate metal-ion uptake by mammalian cells. STEAPs are highly upregulated in several types of cancer, making them potential therapeutic targets. However, the structural basis for STEAP-catalyzed electron transfer through an array of cofactors to metals at the membrane luminal side remains elusive. Here, we report cryo-electron microscopy structures of human STEAP4 in absence and presence of Fe3+-NTA. Domain-swapped, trimeric STEAP4 orients NADPH bound to a cytosolic domain onto axially aligned flavin-adenine dinucleotide (FAD) and a single b-type heme that cross the transmembrane-domain to enable electron transfer. Substrate binding within a positively charged ring indicates that iron gets reduced while in complex with its chelator. These molecular principles of iron reduction provide a basis for exploring STEAPs as therapeutic targets.

Background: PSII is a protein complex that captures sunlight to drive water oxidation. Results: Cyanidioschyzon merolae PSII is protected by reversible reaction center-based non-photochemical quenching. Conclusion: C. merolae PSII employs reaction center non-photochemical quenching as the main photoprotective mechanism. Significance: We provide the first direct evidence of the PSII reaction center as the primary locus of non-photochemical quenching in the extremophilic red algae.

Bacterial cell division is mediated by a multi-protein machine known as the “divisome”, which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.

Lifeact is a novel probe that labels actin filaments in a wide range of organisms. We compared the localization and reorganization of Lifeact:Venus-labeled actin filaments in Arabidopsis root hairs and root epidermal cells of lines that express different levels of Lifeact: Venus with that of actin filaments labeled with GFP:FABD2, a commonly used probe in plants. Unlike GFP:FABD2, Lifeact:Venus labeled the highly dynamic fine F-actin in the subapical region of tip-growing root hairs. Lifeact:Venus expression at varying levels was not observed to affect plant development. However, at expression levels comparable to those of GFP:FABD2 in a well-characterized marker line, Lifeact:Venus reduced reorganization rates of bundles of actin filaments in root epidermal cells. Reorganization rates of cytoplasmic strands, which reflect the reorganization of the actin cytoskeleton, were also reduced in these lines. Moreover, in the same line, Lifeact:Venus-decorated actin filaments were more resistant to depolymerization by latrunculin B than those in an equivalent GFP:FABD2-expressing line. In lines where Lifeact: Venus is expressed at lower levels, these effects are less prominent or even absent. We conclude that Lifeact: Venus reduces remodeling of the actin cytoskeleton in Arabidopsis in a concentration-dependent manner. Since this reduction occurs at expression levels that do not cause defects in plant development, selection of normally growing plants is not sufficient to determine optimal Lifeact expression levels. When correct expression levels of Lifeact have been determined, it is a valuable probe that labels dynamic populations of actin filaments such as fine F-actin, better than FABD2 does.

Membrane contact sites are recognized across eukaryotic systems as important nanostructures. Endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (EPCS) are involved in excitation-contraction coupling, signaling, and plant responses to stress. In this report, we perform a multiscale structural analysis of Arabidopsis EPCS that combines live cell imaging, quantitative transmission electron microscopy (TEM) and electron tomography over a developmental gradient. To place EPCS in the context of the entire cortical ER, we examined green fluorescent protein (GFP)-HDEL in living cells over a developmental gradient, then Synaptotagmin1 (SYT1)-GFP was used as a specific marker of EPCS. In all tissues examined, young, rapidly elongating cells showed lamellar cortical ER and higher density of SYT1-GFP puncta, while in mature cells the cortical ER network was tubular, highly dynamic and had fewer SYT1-labeled puncta. The higher density of EPCS in young cells was verified by quantitative TEM of cryo-fixed tissues. For all cell types, the size of each EPCS had a consistent range in length along the PM from 50 to 300 nm, with microtubules and ribosomes excluded from the EPCS. The structural characterization of EPCS in different plant tissues, and the correlation of EPCS densities over developmental gradients illustrate how ER-PM communication evolves in response to cellular expansion.

Aggregation of the Tau protein into fibrils defines progression of neurodegenerative diseases, including Alzheimer's Disease. The molecular basis for potentially toxic reactions of Tau aggregates is poorly understood. Here we show that π-stacking by Arginine side-chains drives protein binding to Tau fibrils. We mapped an aggregation-dependent interaction pattern of Tau. Fibrils recruit specifically aberrant interactors characterised by intrinsically disordered regions of atypical sequence features. Arginine residues are key to initiate these aberrant interactions. Crucial for scavenging is the guanidinium group of its side chain, not its charge, indicating a key role of π-stacking chemistry for driving aberrant fibril interactions. Remarkably, despite the non-hydrophobic interaction mode, the molecular chaperone Hsp90 can modulate aberrant fibril binding. Together, our data present a molecular mode of action for derailment of protein-protein interaction by neurotoxic fibrils.