Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation.

Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate-binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.

TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation.

The TSC complex is a critical negative regulator of the small GTPase Rheb and mTORC1 in cellular stress signaling. The TSC2 subunit contains a catalytic GTPase activating protein domain and interacts with multiple regulators, while the precise function of TSC1 is unknown. Here we provide a structural characterization of TSC1 and define three domains: a C-terminal coiled-coil that interacts with TSC2, a central helical domain that mediates TSC1 oligomerization, and an N-terminal HEAT repeat domain that interacts with membrane phosphatidylinositol phosphates (PIPs). TSC1 architecture, oligomerization, and membrane binding are conserved in fungi and humans. We show that lysosomal recruitment of the TSC complex and subsequent inactivation of mTORC1 upon starvation depend on the marker lipid PI3,5P2, demonstrating a role for lysosomal PIPs in regulating TSC complex and mTORC1 activity via TSC1. Our study thus identifies a vital role of TSC1 in TSC complex function and mTORC1 signaling.

Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state.

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.

SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM.

Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets-typically comprising thousands of particles-is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200-2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE.

Direct imaging of APP proteolysis in living cells.

Alzheimer's disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aß peptide is widely accepted as being one of the main key events triggering the development of Alzheimer's disease. Aß peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the ß-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α- and the ß-secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α-secretase cleavage without perturbing the ß-secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

Bifunctional Inhibitors as a New Tool To Reduce Cancer Cell Invasion by Impairing MMP-9 Homodimerization.

Protein homodimers play important roles in physiological and pathological processes, including cancer invasion and metastasis. Recently, MMP-9 natural homodimerization via the PEX domain has been correlated with high migration rates of aggressive cancer cells. Here we propose that bifunctional MMP-9 inhibitors designed to impair natural MMP-9 homodimerization promoted by PEX-PEX interactions might be an effective tool to fight cancer cell invasion. Elaborating a previously described dimeric hydroxamate inhibitor 1, new ligands were synthesized with different linker lengths and branch points. Evaluation of the modified bifunctional ligands by X-ray crystallography and biological assays showed that 7 and 8 could reduce invasion in three glioma cell lines expressing MMP-9 at different levels. To rationalize these results, we present a theoretical model of full-length MMP-9 in complex with 7. This pioneering study suggests that a new approach using MMP-9 selective bifunctional inhibitors might lead to an effective therapy to reduce cancer cell invasion.

Crystallization of bi-functional ligand protein complexes.

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects.