We aspired to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, as tools for selectively attaching spectroscopic probes and other functional elements to recombinant proteins. Several supramolecular entities containing 2-4 nitrilotriacetic acid (NTA) moieties were synthesized, which additionally contained an amino group, to which fluorescein was coupled as a sensitive reporter probe. These multivalent chelator heads (MCH) (termed bis-, tris-, and tetrakis-NTA) were characterized with respect to their interaction with hexahistidine (H6)- and decahistidine (H10)-tagged targets. Substantially increased binding stability with increasing number of NTA moieties was observed by analytical size exclusion chromatography. The binding enthalpies as determined by isothermal titration calorimetry increased nearly additively with the number of possible coordinative bonds between chelator heads and tags. Yet, a substantial excess of histidines in the oligohistidine tag was required for obtaining fully additive binding enthalpies. Dissociation kinetics of MCH/oligohistidine complexes measured by fluorescence dequenching showed an increase in stability by 4 orders of magnitude compared to that of mono-NTA, and subnanomolar affinity was reached for tris-NTA. The gain in free energy with increasing multivalency was accompanied by an increasing loss of entropy, which was ascribed to the high flexibility of the binding partners. Numerous applications of these MCHs for noncovalent, high affinity, yet reversible tethering of spectroscopic probes and other functional elements to the recombinant proteins can be envisioned.

During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs interact with membranes and are required for budding processes directed away from the cytosol, including the budding of intralumenal vesicles to form multivesicular bodies, for the budding of some enveloped viruses, and for daughter cell scission in cytokinesis. Here we found that the ESCRT-III proteins CHMP2A and CHMP3 could assemble in vitro into helical tubular structures that expose their membrane interaction sites on the outside of the tubule while the AAA-type ATPase VPS4 could bind on the inside of the tubule and disassemble the tubes upon ATP hydrolysis. CHMP2A and CHMP3 co-polymerized in solution and their membrane targeting was cooperatively enhanced on planar lipid bilayers. Such helical CHMP structures could thus assemble within the neck of an inwardly-budding vesicle, catalyzing late steps in budding under the control of VPS4.

Summary Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human IFN variants signal through the same cell surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique amongst the cytokine receptor superfamily, but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor-points" interspersed amongst ligand-specific interactions that ‘tune’ the relative IFN binding affinities, in an apparent extracellular ‘ligand proofreading’ mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.

Wnts modulate cell proliferation, differentiation and stem cell self-renewal, by inducing β-catenin dependent signaling through Frizzled (Fzd) and Lrp5/6 to regulate cell fate decisions, and the growth and repair of a multitude of tissues1. The 19 mammalian Wnts interact promiscuously with the 10 Fzds, which has complicated the attribution of specific Fzd/Wnt subtype interactions to distinct biological functions. Furthermore, Wnts are post-translationally modified by palmitoylation, which is essential for Wnt secretion and functions as a critical site of interaction with Fzd 2–4. As a result of their acylation, Wnts are very hydrophobic proteins requiring detergents for purification, which presents major obstacles for the preparation and application of recombinant Wnts. This has hindered the delineation of the molecular mechanisms of Wnt signaling activation, understanding of the functional significance of Fzd subtypes, and the use of Wnts as therapeutics. Here we developed surrogate Wnt agonists, water-soluble Fzd-Lrp5/6 heterodimerizers, consisting of Fzd5/8-specific and broadly Fzd-reactive binding domains, that elicit a characteristic β-catenin signaling response in a Fzd-selective fashion, enhance osteogenic lineage commitment of primary mesenchymal stem cells (MSCs), and support the growth of a broad range of primary human organoid cultures comparably to Wnt3a. Furthermore, we demonstrate that the surrogates can be systemically expressed and exhibit Wnt activity in vivo, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signaling can be activated simply through bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced non-lipidated Wnt surrogate agonists offer a new avenue to facilitate functional studies of Wnt signaling and the exploration of Wnt agonists for translational applications in regenerative medicine.

Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals where it can be both a commensal and a pathogen1–3. Intricate regulatory mechanisms ensure bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat4,5. Yet no mechanism is known for replacing OMPs in the outer membrane (OM), a biological enigma further confounded by the lack of an energy source and the high stability6 and abundance of OMPs5. Here, we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature wherein old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics (MD) simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form islands of ~0.5 μm diameter where their diffusion was restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the OM7,8. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence the OM of a Gram-negative bacterium is a spatially and temporally organised structure and this organisation lies at the heart of how OMPs are turned over in the membrane.

Alpha and beta interferons (IFN-␣Type I interferons (IFNs) form a family of multifunctional cytokines initially described for their direct antiviral effect but now also recognized as major elements of the immune response (19,46). Differential activities of IFN subtypes have been reported (3) and used in the clinic for the treatment of various pathologies, including viral hepatitis (IFN-␣2) and multiple sclerosis (IFN-␤) (32). All type I IFNs are recognized by a single shared receptor composed of two transmembrane proteins, IFNAR1 and IFNAR2. Because of the much faster k on and much slower k off of IFN-␣2 towards IFNAR2 than those measured for IFNAR1 (18,34,40), a two-step assembling mechanism was proposed for the interaction between IFN and the two receptors (Fig. 1B). After binding of IFN-␣2 to IFNAR2 (k a1 ), IFNAR1 transiently associates in a second step to the complex (k a2 ) (18,25). Owing to the short lifetime of the IFN-␣2-IFNAR1 interaction, the complex dissociates (k d2 ) and reassociates (k a2 ) in a fast manner. Thus, depending on the receptor surface concentrations and ligand binding affinities, only part of the bound ligand is involved in the active ternary complex.After formation of the ternary complex, the interferon signal is transduced through the receptor-associated JAK kinases, with the STAT transcription factors as their main targets (3).

SUMMARY Most cell surface receptors for cytokines and growth factors signal as dimers, but it is unclear if remodeling receptor dimer topology is a viable strategy to ‘tune’ signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitude varied from full to minimal agonism, and structures of the DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased, or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.

Summary Type I interferons (IFNs) form a network of homologous cytokines that bind to a shared, heterodimeric cell surface receptor and engage signaling pathways that activate innate and adaptive immune responses. The ability of IFNs to mediate differential responses through the same cell surface receptor has been subject of a controversial debate and has important medical implications. During the past decade, a comprehensive insight into the structure, energetics, and dynamics of IFN recognition by its two-receptor subunits, as well as detailed correlations with their functional properties on the level of signal activation, gene expression, and biological responses were obtained. All type I IFNs bind the two-receptor subunits at the same sites and form structurally very similar ternary complexes. Differential IFN activities were found to be determined by different lifetimes and ligand affinities toward the receptor subunits, which dictate assembly and dynamics of the signaling complex in the plasma membrane. We present a simple model, which explains differential IFN activities based on rapid endocytosis of signaling complexes and negative feedback mechanisms interfering with ternary complex assembly. More insight into signaling pathways as well as endosomal signaling and trafficking will be required for a comprehensive understanding, which will eventually lead to therapeutic applications of IFNs with increased efficacy.