Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments.

The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

Protein tyrosine phosphatase SHP2/PTPN11 mistargeting as a consequence of SH2-domain point mutations associated with Noonan Syndrome and leukemia.

SHP2/PTPN11 is a key regulator of cytokine, growth factor and integrin signaling. SHP2 influences cell survival, proliferation and differentiation by regulating major signaling pathways. Mutations in PTPN11 cause severe diseases like Noonan, LEOPARD syndrome or leukemia. Whereas several of these mutations result in altered enzymatic activity due to impaired auto-inhibition, not all disease patterns can be explained by this mechanism. In this study we analyzed altered binding properties of disease-related SHP2-mutants bearing point mutations within the SH2-domain (T42A, E139D, and R138Q). Mutants were chosen according to SPR assays, which revealed different binding properties of mutated SH2 towards phosphorylated receptor peptides. To analyze global changes in mutant binding properties we applied quantitative mass spectrometry (SILAC). Using an in vitro approach we identified overall more than 1000 protein candidates, which specifically bind to the SH2-domain of SHP2. We discovered that mutations in the SH2-domain selectively affected protein enrichment by altering the binding capacity of the SH2-domain. Mutation-dependent, enhanced or reduced exposure of SHP2 to its binding partners could have an impact on the dynamics of signaling networks. Thus, disease-associated mutants of SHP2 should not only be discussed in the context of deregulated auto-inhibition but also with respect to deregulated protein targeting of the SHP2 mutants. BIOLOGICAL SIGNIFICANCE: Using quantitative mass spectrometry based proteomics we provided evidence that disease related mutations in SHP2 domains of SHP2 are able to influence SHP2 recruitment to its targets in mutation dependent manner. We discovered that mutations in the SH2-domain selectively affected protein enrichment ratios suggesting altered binding properties of the SH2-domain. We demonstrated that mutations within SHP2, which had been attributed to affect the enzymatic activity (i.e. affect the open/close status of SHP2), also differ in respect to binding properties. Our study indicates that SHP2 mutations need to be discussed not only in terms of deregulated auto-inhibition but also with respect to deregulated protein targeting properties of the SHP2 mutants. Discovery of the new binding partners for disease-related SHP2 mutants might provide a fruitful foundation for developing strategies targeting Noonan-associated leukemia.

Spatial organization of lipid phases in micropatterned polymer-supported membranes.

We have established an approach for the spatial control of lipid phase separation in tethered polymer-supported membranes (PSMs), which were obtained by vesicle fusion on a poly(ethylene glycol) polymer brush functionalized with fatty acid moieties. Phase separation of ternary lipid mixtures (1,2-dioleoyl-sn-glycero-3-phosphocholine/sphingomyelin/cholesterol) into liquid-disordered (l(d)) and liquid-ordered (l(o)) phases within both leaflets was obtained with palmitic acid as the anchoring group. In contrast, tethering of the PSM with oleic acid interfered with the phase separation in the surface-proximal leaflet. We exploited this feature for the assembly of l(o) domains within PSMs into defined structures by binary micropatterning of palmitic and oleic acid into complementary areas. Ternary lipid mixtures spontaneously separated into l(o) and l(d) phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSMs strictly partitioned into the l(d) phase. Hence, the l(o) phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains.

Reconstitution of membrane proteins into polymer-supported membranes for probing diffusion and interactions by single molecule techniques.

We have established a robust and versatile analytical platform for probing membrane protein function in a defined lipid environment on solid supports. This approach is based on vesicle capturing onto an ultrathin poly(ethylene glycol) (PEG) polymer brush functionalized with fatty acid moieties and subsequent vesicle fusion into a contiguous membrane. In order to ensure efficient formation of these tethered polymer-supported membranes (PSM), very small unilamellar vesicles (VSUV) containing fluorescent lipids or model transmembrane proteins were generated by detergent depletion with cyclodextrin. Thus, very rapid reconstitution of membrane proteins into PSM was possible in a format compatible with microfluidics. Moreover, surfaces could be regenerated with detergent solution and reused multiple times. Lipid and protein diffusion in these membranes was investigated by fluorescence recovery after photobleaching, single molecule tracking, and fluorescence correlation spectroscopy. Full mobility of lipids and a high degree of protein mobility as well as homogeneous diffusion of both were observed. Quantitative ligand binding studies by solid phase detection techniques confirmed functional integrity of a transmembrane receptor reconstituted into these PSM. Colocomotion of individual ligand-receptor complexes was detected, demonstrating the applicability for single molecule fluorescence techniques.

Functional immobilization and patterning of proteins by an enzymatic transfer reaction.

Functional immobilization and lateral organization of proteins into micro- and nanopatterns is an important prerequisite for miniaturizing bioanalytical and biotechnological devices. Here, we report an approach for efficient site-specific protein immobilization based on enzymatic phosphopantetheinyl transfer (PPT) from coenzyme A (CoA)-functionalized glass-type surfaces to specific peptide tags. We devised a bottom-up surface modification approach for coupling CoA densely to a molecular poly(ethylene glycol) polymer brush. Site-specific enzymatic immobilization of proteins fused to different target peptides for the PPTase Sfp was confirmed by real-time label-free detection. Quantitative protein-protein interaction experiments confirmed that significantly more than 50% of the immobilized protein was fully active. The method was successfully applied with different proteins. However, different immobilization efficiencies of PPT-based immobilization were observed for different peptide tags being fused to the N- and C-termini of proteins. On the basis of this immobilization method, we established photolithographic patterning of proteins into functional binary microstructures.

Structure of Aquifex aeolicus argonaute highlights conformational flexibility of the PAZ domain as a potential regulator of RNA-induced silencing complex function.

Gene silencing mediated by RNA interference requires the sequence-specific recognition of target mRNA by the endonuclease Argonaute, the primary enzymatic component of the RNA-induced silencing complex. We report the crystal structure of Aquifex aeolicus Argonaute, refined at 3.2A resolution. Relative to recent Argonaute structures, a 24 degrees reorientation of the PAZ domain in our structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. This rearrangement leads to a branched, Y-shaped system of grooves that extends through the molecule and merges in a central channel containing the catalytic residues. A 5.5-ns molecular dynamics simulation of Argonaute shows a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute monitored by total internal reflection fluorescence spectroscopy shows biphasic kinetics, also indicative of domain rearrangement upon DNA binding. Conformational rearrangement of the PAZ domain may therefore be critical for the catalytic cycle of Argonaute and the RNA-induced silencing complex.