ColabSeg: An interactive tool for editing, processing, and visualizing membrane segmentations from cryo-ET data.

Cellular cryo-electron tomography (cryo-ET) has emerged as a key method to unravel the spatial and structural complexity of cells in their near-native state at unprecedented molecular resolution. To enable quantitative analysis of the complex shapes and morphologies of lipid membranes, the noisy three-dimensional (3D) volumes must be segmented. Despite recent advances, this task often requires considerable user intervention to curate the resulting segmentations. Here, we present ColabSeg, a Python-based tool for processing, visualizing, editing, and fitting membrane segmentations from cryo-ET data for downstream analysis. ColabSeg makes many well-established algorithms for point-cloud processing easily available to the broad community of structural biologists for applications in cryo-ET through its graphical user interface (GUI). We demonstrate the usefulness of the tool with a range of use cases and biological examples. Finally, for a large Mycoplasma pneumoniae dataset of 50 tomograms, we show how ColabSeg enables high-throughput membrane segmentation, which can be used as valuable training data for fully automated convolutional neural network (CNN)-based segmentation.

Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMß2.

The integrin receptor αMß2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of αMß2 signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human αMß2 headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H αM variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside-inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the αM subunit in an Mg2+-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the αM subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound αMß2 with iC3b, suggesting that it may represent a novel high-affinity proteinaceous αMß2-specific agonist. Taken together, our data suggest that the iC3b-αMß2 complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to αMß2.

Novel homozygous CD46 variant with C-isoform expression affects C3b inactivation in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy that may lead to organ failure. Dysregulation of the complement system can cause aHUS, and various disease-related variants in the complement regulatory protein CD46 are described. We here report a pediatric patient with aHUS carrying a hitherto unreported homozygous variant in CD46 (NM_172359.3:c.602C>T p.(Ser201Leu)). In our functional analyses, this variant caused complement dysregulation through three separate mechanisms. First, CD46 surface expression on the patient's blood cells was significantly reduced. Second, stably expressing CD46(Ser201Leu) cells bound markedly less to patterns of C3b than CD46 WT cells. Third, the patient predominantly expressed the rare isoforms of CD46 (C dominated) instead of the more common isoforms (BC dominated). Using BC1 and C1 expressing cell lines, we found that the C1 isoform bound markedly less C3b than the BC1 isoform. These results highlight the coexistence of multiple mechanisms that may act synergistically to disrupt CD46 function during aHUS development.

Complement Receptor 3 Forms a Compact High-Affinity Complex with iC3b.

Complement receptor 3 (CR3, also known as Mac-1, integrin αMß2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment iC3b and, to a lesser extent, C3dg. Although the interaction between the iC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-iC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between iC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for iC3b compared with 515 nM for the iC3b thioester domain, whereas experiments monitoring binding of iC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-iC3b interaction. Small angle x-ray scattering analysis revealed that iC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, iC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the iC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the iC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by iC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.

A Complement C3-Specific Nanobody for Modulation of the Alternative Cascade Identifies the C-Terminal Domain of C3b as Functional in C5 Convertase Activity.

The complement system is an intricate cascade of the innate immune system and plays a key role in microbial defense, inflammation, organ development, and tissue regeneration. There is increasing interest in developing complement regulatory and inhibitory agents to treat complement dysfunction. In this study, we describe the nanobody hC3Nb3, which is specific for the C-terminal C345c domain of human and mouse complement component C3/C3b/C3c and potently inhibits C3 cleavage by the alternative pathway. A high-resolution structure of the hC3Nb3-C345c complex explains how the nanobody blocks proconvertase assembly. Surprisingly, although the nanobody does not affect classical pathway-mediated C3 cleavage, hC3Nb3 inhibits classical pathway-driven hemolysis, suggesting that the C-terminal domain of C3b has an important function in classical pathway C5 convertase activity. The hC3Nb3 nanobody binds C3 with low nanomolar affinity in an SDS-resistant complex, and the nanobody is demonstrated to be a powerful reagent for C3 detection in immunohistochemistry and flow cytometry. Overall, the hC3Nb3 nanobody represents a potent inhibitor of both the alternative pathway and the terminal pathway, with possible applications in complement research, diagnostics, and therapeutics.

A C3-specific nanobody that blocks all three activation pathways in the human and murine complement system.

The complement system is a tightly controlled proteolytic cascade in the innate immune system, which tags intruding pathogens and dying host cells for clearance. An essential protein in this process is complement component C3. Uncontrolled complement activation has been implicated in several human diseases and disorders and has spurred the development of therapeutic approaches that modulate the complement system. Here, using purified proteins and several biochemical assays and surface plasmon resonance, we report that our nanobody, hC3Nb2, inhibits C3 deposition by all complement pathways. We observe that the hC3Nb2 nanobody binds human native C3 and its degradation products with low nanomolar affinity and does not interfere with the endogenous regulation of C3b deposition mediated by Factors H and I. Using negative stain EM analysis and functional assays, we demonstrate that hC3Nb2 inhibits the substrate-convertase interaction by binding to the MG3 and MG4 domains of C3 and C3b. Furthermore, we notice that hC3Nb2 is cross-reactive and inhibits the lectin and alternative pathway in murine serum. We conclude that hC3Nb2 is a potent, general, and versatile inhibitor of the human and murine complement cascades. Its cross-reactivity suggests that this nanobody may be valuable for analysis of complement activation within animal models of both acute and chronic diseases.

Functional and structural insight into properdin control of complement alternative pathway amplification.

Properdin (FP) is an essential positive regulator of the complement alternative pathway (AP) providing stabilization of the C3 and C5 convertases, but its oligomeric nature challenges structural analysis. We describe here a novel FP deficiency (E244K) caused by a single point mutation which results in a very low level of AP activity. Recombinant FP E244K is monomeric, fails to support bacteriolysis, and binds weakly to C3 products. We compare this to a monomeric unit excised from oligomeric FP, which is also dysfunctional in bacteriolysis but binds the AP proconvertase, C3 convertase, C3 products and partially stabilizes the convertase. The crystal structure of such a FP-convertase complex suggests that the major contact between FP and the AP convertase is mediated by a single FP thrombospondin repeat and a small region in C3b. Small angle X-ray scattering indicates that FP E244K is trapped in a compact conformation preventing its oligomerization. Our studies demonstrate an essential role of FP oligomerization in vivo while our monomers enable detailed structural insight paving the way for novel modulators of complement.

Structure of intact IgE and the mechanism of ligelizumab revealed by electron microscopy.

BACKGROUND: IgE is the central antibody isotype in TH2-biased immunity and allergic diseases. The structure of intact IgE and the impact of IgE-targeting molecules on IgE however remain elusive. In order to obtain insights into IgE biology and the clinical impact, we aimed for structure determination of IgE and the complex of IgE with the anti-IgE antibody ligelizumab. METHODS: Structures of two distinct intact IgE with specificity for cross-reactive carbohydrate determinants and Der p 2 as well as complexes of ligelizumab-Fab with IgE and IgE Fc were assessed by negative stain electron microscopy and solution scattering. Inhibition of IgE binding and displacement of receptor-bound IgE were assessed using cellular assays, basophil activation testing and ELIFAB assays. RESULTS: Our data reveal that the investigated IgE molecules share an overall rigid conformation. In contrast to the IgE Fc fragment, the IgE Fc in intact IgE is significantly less asymmetrically bent. The proximal and the distal Fabs are rigidly tethered to the Fc. Binding of ligelizumab to IgE in a 2:1 stoichiometry induces an extended and twofold symmetrical conformation of IgE, which retains a rigid Fab-Fc architecture. Analyses of effector cell activation revealed that ligelizumab inhibits IgE binding without displacing receptor-bound IgE. Together with an interference of CD23 binding, the data underline a functional activity similar to omalizumab. CONCLUSIONS: Our data reveal the first structures of intact IgE suggesting that the IgE Fab is fixed relative to the Fc. Furthermore, we provide a structural rationale for the inhibitory mechanism of ligelizumab.

Structure and activation of C1, the complex initiating the classical pathway of the complement cascade.

The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition molecule C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific molecular patterns such as IgG- and IgM-containing immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramolecular reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, we show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. Our structural studies indicate that the C1 complex adopts a conformation incompatible with intramolecular activation of C1, suggesting instead that intermolecular proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. Our results rationalize how a multitude of structurally unrelated molecular patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.

A potent complement factor C3-specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement.

The complement system is a complex, carefully regulated proteolytic cascade for which suppression of aberrant activation is of increasing clinical relevance, and inhibition of the complement alternative pathway is a subject of intense research. Here, we describe the nanobody hC3Nb1 that binds to multiple functional states of C3 with subnanomolar affinity. The nanobody causes a complete shutdown of alternative pathway activity in human and murine serum when present in concentrations comparable with that of C3, and hC3Nb1 is shown to prevent proconvertase assembly, as well as binding of the C3 substrate to C3 convertases. Our crystal structure of the C3b-hC3Nb1 complex and functional experiments demonstrate that proconvertase formation is blocked by steric hindrance between the nanobody and an Asn-linked glycan on complement factor B. In addition, hC3Nb1 is shown to prevent factor H binding to C3b, rationalizing its inhibition of factor I activity. Our results identify hC3Nb1 as a versatile, inexpensive, and powerful inhibitor of the alternative pathway in both human and murine in vitro model systems of complement activation.

ITIH4 acts as a protease inhibitor by a novel inhibitory mechanism.

Inter-α-inhibitor heavy chain 4 (ITIH4) is a poorly characterized plasma protein that is proteolytically processed in multiple pathological conditions. However, no biological function of ITIH4 has been identified. Here, we show that ITIH4 is cleaved by several human proteases within a protease-susceptible region, enabling ITIH4 to function as a protease inhibitor. This is exemplified by its inhibition of mannan-binding lectin-associated serine protease-1 (MASP-1), MASP-2, and plasma kallikrein, which are key proteases for intravascular host defense. Mechanistically, ITIH4 acts as bait that, upon cleavage, forms a noncovalent, inhibitory complex with the executing protease that depends on the ITIH4 von Willebrand factor A domain. ITIH4 inhibits the MASPs by sterically preventing larger protein substrates from accessing their active sites, which remain accessible and fully functional toward small substrates. Thus, we demonstrate that ITIH4 functions as a protease inhibitor by a previously undescribed inhibitory mechanism.

Structural Basis for Properdin Oligomerization and Convertase Stimulation in the Human Complement System.

Properdin (FP) is a positive regulator of the immune system stimulating the activity of the proteolytically active C3 convertase C3bBb in the alternative pathway of the complement system. Here we present two crystal structures of FP and two structures of convertase bound FP. A structural core formed by three thrombospondin repeats (TSRs) and a TB domain harbors the convertase binding site in FP that mainly interacts with C3b. Stabilization of the interaction between the C3b C-terminus and the MIDAS bound Mg2+ in the Bb protease by FP TSR5 is proposed to underlie FP convertase stabilization. Intermolecular contacts between FP and the convertase subunits suggested by the structure were confirmed by binding experiments. FP is shown to inhibit C3b degradation by FI due to a direct competition for a common binding site on C3b. FP oligomers are held together by two sets of intermolecular contacts, where the first is formed by the TB domain from one FP molecule and TSR4 from another. The second and largest interface is formed by TSR1 and TSR6 from the same two FP molecules. Flexibility at four hinges between thrombospondin repeats is suggested to enable the oligomeric, polydisperse, and extended architecture of FP. Our structures rationalize the effects of mutations associated with FP deficiencies and provide a structural basis for the analysis of FP function in convertases and its possible role in pattern recognition.

Trapping IgE in a closed conformation by mimicking CD23 binding prevents and disrupts FcεRI interaction.

Anti-IgE therapeutics interfere with the ability of IgE to bind to its receptors on effector cells. Here we report the crystal structure of an anti-IgE single-domain antibody in complex with an IgE Fc fragment, revealing how the antibody inhibits interactions between IgE and the two receptors FcεRI and CD23. The epitope overlaps only slightly with the FcεRI-binding site but significantly with the CD23-binding site. Solution scattering studies of the IgE Fc reveal that antibody binding induces a half-bent conformation in between the well-known bent and extended IgE Fc conformations. The antibody acts as functional homolog of CD23 and induces a closed conformation of IgE Fc incompatible with FcεRI binding. Notably the antibody displaces IgE from both CD23 and FcεRI, and abrogates allergen-mediated basophil activation and facilitated allergen binding. The inhibitory mechanism might facilitate strategies for the future development of anti-IgE therapeutics for treatment of allergic diseases.

Structure of the omalizumab Fab.

Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor FcℇRI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc Cℇ2-4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Šwere obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.