GPC receptors and not ligands decide the binding mode in neuropeptide Y multireceptor/multiligand system.

Many G protein-coupled receptors belong to families of different receptor subtypes, which are recognized by a variety of distinct ligands. To study such a multireceptor/multiligand system, we investigated the Y-receptor family. This family consists of four G protein-coupled Y receptors in humans (hY 1R, hY 2R, hY 4R, and hY 5R) and is activated by the so-called NPY hormone family, which itself consists of three native peptide ligands named neuropeptide Y (NPY), pancreatic polypeptide (PP), and peptide YY (PYY). The hY 5R shows high affinity for all ligands, although for PP binding, the affinity is slightly decreased. As a rational explanation, we suggest that Tyr (27) is lost as a contact point between PP and the hY 5R in contrast to NPY or PYY. Furthermore, several important residues for ligand binding were identified by the first extensive mutagenesis study of the hY 5R. Using a complementary mutagenesis approach, we were able to discover a novel interaction point between hY 5R and NPY. The interaction between NPY(Arg (25)) and hY 5R(Asp (2.68)) as well as between NPY(Arg (33)) and hY 5R(Asp (6.59)) is maintained in the binding of PYY and PP to hY 5R but different to the PP-hY 4R and NPY-hY 1R contact points. Therefore, we provide evidence that the receptor subtype and not the pre-orientated conformation of the ligand at the membrane decides the binding mode. Furthermore, the first hY 5R model was set up on the basis of the crystal structure of bovine rhodopsin. We can show that most of the residues identified to be critical for ligand binding are located within the now postulated binding pocket.

Development of bispecific molecules for the in situ detection of protein-protein interactions and protein phosphorylation.

Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications.

S100 proteins interact with the N-terminal domain of MDM2.

S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro.

Modulation of the oligomerization state of p53 by differential binding of proteins of the S100 family to p53 monomers and tetramers.

We investigated the ways S100B, S100A1, S100A2, S100A4, and S100A6 bind to the different oligomeric forms of the tumor suppressor p53 in vitro, using analytical ultracentrifugation and multiangle light scattering. It is established that members of the S100 protein family bind to the tetramerization domain (residues 325-355) of p53 when it is uncovered in the monomer, and so binding can disrupt the tetramer. We found a stoichiometry of one dimer of S100 bound to a monomer of p53. We discovered that some S100 proteins could also bind to the tetramer. S100B bound the tetramer and also disrupted the dimer by binding monomeric p53. S100A2 bound monomeric p53 as well as tetrameric, whereas S100A1 only bound monomeric p53. S100A6 bound more tightly to tetrameric than to monomeric p53. We also identified an additional binding site for S100 proteins in the transactivation domain (1-57) of p53. Based on our results and published observations in vivo, we propose a model for the binding of S100 proteins to p53 that can explain both activation and inhibition of p53-mediated transcription. Depending on the concentration of p53 and the member of the S100 family, binding can alter the balance between monomer and tetramer in either direction.

Posttranslational modifications affect the interaction of S100 proteins with tumor suppressor p53.

Proteins of the S100 family bind to the intrinsically disordered transactivation domain (TAD; residues 1-57) and C-terminus (residues 293-393) of the tumor suppressor p53. Both regions provide sites that are subject to posttranslational modifications, such as phosphorylation and acetylation, that can alter the affinity for interacting proteins such as p300 and MDM2. Here, we found that S100A1, S100A2, S100A4, S100A6, and S100B bound to two subdomains of the TAD (TAD1 and TAD2). Both subdomains were mandatory for high-affinity binding to S100 proteins. Phosphorylation of Ser and Thr residues increased the affinity for the p53 TAD. Conversely, acetylation and phosphorylation of the C-terminus of p53 decreased the affinity for S100A2 and S100B. In contrast, we found that nitrosylation of S100B caused a minor increase in binding to the p53 C-terminus, whereas binding to the TAD remained unaffected. As activation of p53 is usually accompanied by phosphorylation and acetylation at several sites, our results suggest that a shift in binding from the C-terminus in favor of the N-terminus occurs upon the modification of p53. We propose that binding to the p53 TAD might be involved in the stimulation of p53 activity by S100 proteins.