Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I ...cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.
We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild ...detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level.
Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network ...therefore controls IFN signaling, which in turn may interfere with medical interventions. The JAK-STAT signaling pathway transmits the IFN extracellular signal to the nucleus, thus resulting in alterations in gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized, crucial component of the USP18-mediated negative-feedback control in both human and mouse cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2-binding site. This mechanistic coupling of effector and negative-feedback functions of STAT2 may provide novel strategies for treatment of IFN-signaling-related human diseases.
Unraveling the spatiotemporal organization of signaling complexes within the context of plasma membrane nanodomains has remained a highly challenging task. Here, we have applied super-resolution ...image correlation based on tracking and localization microscopy (TALM) for probing transient confinement as well as ligand binding and intracellular effector recruitment of the type I interferon (IFN) receptor in the plasma membrane of live cells. Ligand and receptor were labeled with monofunctional quantum dots, thus allowing long-term tracking with very high spatial and temporal resolution without an artificial receptor cross-linking at the cell surface. Dual-color TALM was employed for visualizing protein–protein interactions involved in IFN signaling at both sides of the plasma membrane with high spatial and temporal resolution. By pair correlation analyses based on time-lapse TALM images (pcTALM), complex assembly within dynamic submicroscopic zones was identified. Strikingly, recruitment of the IFN effector protein signal transducer and activator of transcription 2 (STAT2) into these dynamic signaling zones could be observed. The results suggest that confined diffusion zones in the plasma membrane are employed as transient platforms for the assembly of signaling complexes.
Interactions of proteins in the plasma membrane are notoriously challenging to study under physiological conditions. We report in this paper a generic approach for spatial organization of plasma ...membrane proteins into micropatterns as a tool for visualizing and quantifying interactions with extracellular, intracellular, and transmembrane proteins in live cells. Based on a protein-repellent poly(ethylene glycol) polymer brush, micropatterned surface functionalization with the HaloTag ligand for capturing HaloTag fusion proteins and RGD peptides promoting cell adhesion was devised. Efficient micropatterning of the type I interferon (IFN) receptor subunit IFNAR2 fused to the HaloTag was achieved, and highly specific IFN binding to the receptor was detected. The dynamics of this interaction could be quantified on the single molecule level, and IFN-induced receptor dimerization in micropatterns could be monitored. Assembly of active signaling complexes was confirmed by immunostaining of phosphorylated Janus family kinases, and the interaction dynamics of cytosolic effector proteins recruited to the receptor complex were unambiguously quantified by fluorescence recovery after photobleaching.
Monoclonal antibodies (Abs) that recognize major histocompatability complex (MHC)-presented tumor antigens in a manner similar to T cell receptors (TCRs) have great potential as cancer ...immunotherapeutics. However, isolation of 'TCR-mimic' (TCRm) Abs is laborious because Abs have not evolved the structurally nuanced peptide-MHC restriction of αβ-TCRs. Here, we present a strategy for rapid isolation of highly peptide-specific and 'MHC-restricted' Abs by re-engineering preselected Abs that engage peptide-MHC in a manner structurally similar to that of conventional αβ-TCRs. We created structure-based libraries focused on the peptide-interacting residues of TCRm Ab complementarity-determining region (CDR) loops, and rapidly generated MHC-restricted Abs to both mouse and human tumor antigens that specifically killed target cells when formatted as IgG, bispecific T cell engager (BiTE) and chimeric antigen receptor-T (CAR-T). Crystallographic analysis of one selected pMHC-restricted Ab revealed highly peptide-specific recognition, validating the engineering strategy. This approach can yield tumor antigen-specific antibodies in several weeks, potentially enabling rapid clinical translation.
Triggered immobilization of proteins in the plasma membrane of living cells into functional micropatterns is established by using an adaptor protein, which is comprised of an antiGFP nanobody fused ...to the HaloTag protein. Efficient in situ reorganization of the type I interferon receptor subunits as well as intact, fully functional signaling complexes in living cells are achieved by this method.
Functionalization of quantum dots (QDs) was achieved by controlling the surface density of functional groups by electrostatic repulsion. These QDs were conjugated with His‐tagged proteins in vitro ...and in live cells by self‐assembly without requiring further fractionation, and dual‐color tracking of cell surface receptors was possible.
KtrB, the K+-translocating subunit of the Na+-dependent bacterial K+ uptake system KtrAB, consists of four M1PM2 domains, in which M1 and M2 are transmembrane helices and P indicates a p-loop that ...folds back from the external medium into the cell membrane. The transmembrane stretch M2C is, with its 40 residues, unusually long. It consists of three parts, the hydrophobic helices M2C1 and M2C3, which are connected by a nonhelical M2C2 region, containing conserved glycine, alanine, serine, threonine, and lysine residues. Several point mutations in M2C2 led to a huge gain of function of K+ uptake by KtrB from the bacterium Vibrio alginolyticus. This effect was exclusively due to an increase in Vmax for K+ transport. Na+ translocation by KtrB was not affected. Partial to complete deletions of M2C2 also led to enhanced Vmax values for K+ uptake via KtrB. However, several deletion variants also exhibited higher Km values for K+ uptake and at least one deletion variant, KtrBΔ326–328, also transported Na+ faster. The presence of KtrA did not suppress any of these effects. For the deletion variants, this was due to a diminished binding of KtrA to KtrB. PhoA studies indicated that M2C2 forms a flexible structure within the membrane allowing M2C3 to be directed either to the cytoplasm or (artificially) to the periplasm. These data are interpreted to mean (i) that region M2C2 forms a flexible gate controlling K+ translocation at the cytoplasmic side of KtrB, and (ii) that M2C2 is required for the interaction between KtrA and KtrB.
At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light ...chain beta-2 microglobulin (β2m, encoded by B2M), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single-molecule co-tracking. We identify non-covalent MHC-I FHC dimers, with dimerization mediated by the α3 domain, as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single-molecule colocalization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to that seen for β2m.
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