The binding affinity of PTPN13's tandem PDZ2/3 domain is allosterically modulated.

BACKGROUND: Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, is a large multi-domain non-transmembrane scaffolding protein with a molecular mass of 270 kDa. It is involved in the regulation of several cellular processes such as cytokinesis and actin-cytoskeletal rearrangement. The modular structure of PTPN13 consists of an N-terminal KIND domain, a FERM domain, and five PDZ domains, followed by a C-terminal protein tyrosine phosphatase domain. PDZ domains are among the most abundant protein modules and they play a crucial role in signal transduction of protein networks. RESULTS: Here, we have analysed the binding characteristics of the isolated PDZ domains 2 and 3 from PTPN13 and compared them to the tandem domain PDZ2/3, which interacts with 12 C-terminal residues of the tumour suppressor protein of APC, using heteronuclear multidimensional NMR spectroscopy. Furthermore, we could show for the first time that PRK2 is a weak binding partner of PDZ2 and we demonstrate that the presence of PDZ3 alters the binding affinity of PDZ2 for APC, suggesting an allosteric effect and thereby modulating the binding characteristics of PDZ2. A HADDOCK-based molecular model of the PDZ2/3 tandem domain from PTPN13 supports these results. CONCLUSIONS: Our study of tandem PDZ2/3 in complex with APC suggests that the interaction of PDZ3 with PDZ2 induces an allosteric modulation within PDZ2 emanating from the back of the domain to the ligand binding site. Thus, the modified binding preference of PDZ2 for APC could be explained by an allosteric effect and provides further evidence for the pivotal function of PDZ2 in the PDZ123 domain triplet within PTPN13.

Phosphorylation orchestrates the structural ensemble of the intrinsically disordered protein HMGA1a and modulates its DNA binding to the NFκB promoter.

High Mobility Group Protein A1a (HMGA1a) is a highly abundant nuclear protein, which plays a crucial role during embryogenesis, cell differentiation, and neoplasia. Here, we present the first ever NMR-based structural ensemble of full length HMGA1a. Our results show that the protein is not completely random coil but adopts a compact structure consisting of transient long-range contacts, which is regulated by post-translational phosphorylation. The CK2-, cdc2- and cdc2/CK2-phosphorylated forms of HMGA1a each exhibit a different binding affinity towards the PRD2 element of the NFκB promoter. Our study identifies connected regions between phosphorylation sites in the wildtype ensemble that change considerably upon phosphorylation, indicating that these posttranslational modifications sites are part of an electrostatic contact network that alters the structural ensemble by shifting the conformational equilibrium. Moreover, ITC data reveal that the CK2-phosphorylated HMGA1a exhibits a different DNA promoter binding affinity for the PRD2 element. Furthermore, we present the first structural model for AT-hook 1 of HMGA1a that can adopt a transient α-helical structure, which might serve as an additional regulatory mechanism in HMAG1a. Our findings will help to develop new therapeutic strategies against HMGA1a-associated cancers by taking posttranslational modifications into consideration.

Bax to the future - A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies.

Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.

The Structure in Solution of Fibronectin Type III Domain 14 Reveals Its Synergistic Heparin Binding Site.

Fibronectin is a large multidomain protein of the extracellular matrix that harbors two heparin binding sites, Hep-I and Hep-II, which support the heparin-dependent adhesion of melanoma and neuroblastoma cells [Barkalow, F. J. B., and Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818; McCarthy, J. B., et al. (1988) Biochemistry 27, 1380-1388; Drake, S. L., et al. (1993) J. Biol. Chem. 268, 15859-15867]. The stronger heparin/HS binding site on fibronectin, Hep-II, spans fibronectin type III domains 12-14. Previous site-directed mutagenesis, nuclear magnetic resonance (NMR) chemical shift perturbation, and crystallographic structural studies all agree that the main heparin binding site is located on the surface of fibronectin type III domain 13 [Ingham, K. C., et al. (1993) Biochemistry 32, 12548-12553; Sharma, A., et al. (1999) EMBO J. 18, 1468-1479; Sachchidanand, L. O., et al. (2002) J. Biol. Chem. 277, 50629-50635]. However, the "synergy site" for heparin binding located on fibronectin type III domain 14 remained elusive because the actual binding sites could not be identified. Using NMR spectroscopy and isothermal titration calorimetry, we show here that heparin is able to bind to a cationic 'cradle' of fibronectin type III domain 14 formed by the PRARI sequence, which is involved in the integrin α4ß1 interaction [Mould, A. P., and Humphries, M. J. (1991) EMBO J. 10, 4089-4095], and to the flexible loop comprising residues KNNQKSE between the last two ß-strands, D and E, of FN14. Our data reveal that the individual FN14 domain binds to the sulfated sugars Dp8 and Reviparin with affinities similar to those of the individual domain FN13 [Breddin, H. K. (2002) Expert Opin. Pharmacother. 3, 173-182]. It is noteworthy that by introduction of the last ß-strand of FN13 and the linker region between FN type III domains 13 and 14, the perturbation of NMR chemical shifts by heparin is significantly reduced, especially at the PRARI site. This indicates that the Hep-II binding site of fibronectin is mainly located on FN13 and the synergistic binding site on FN14 involves only the KNNQKSE sequence.

Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers.

The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H-Ras, N-Ras, and K-Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best-known member and has gained significant scientific as well as public attention as an endocrine disrupting chemical, a fact that eventually led to its replacement. However, compounds used to replace BPA still contain the molecular scaffold of bisphenols. BPA, BPAF, BPB, BPE, BPF, and an amine-substituted BPAF-derivate all interact with all GDP-bound Ras-Isoforms through binding to a common site on these proteins. NMR-, SOScat-, and GDI- assay-based data revealed a new bisphenol-induced, allosterically activated GDP-bound Ras conformation that define these plasticisers as Ras allosteric agonists.

Human melanoma inhibitory protein binds to the FN12-14 Hep II domain of fibronectin.

The heparin binding site (Hep II) of fibronectin plays a major role in tumor cell metastasis. Its interaction with heparan sulfate proteoglycans occurs in a variety of physiological processes including focal adhesion and migration. The melanoma inhibitory activity (MIA) is an important protein that is functionally involved in melanoma development, progression, and tumor cell invasion. After its secretion by malignant melanoma cells, MIA interacts with fibronectin and thereby actively facilitates focal cell detachment from surrounding structures and strongly promotes tumor cell invasion and the formation of metastases. In this report, the authors have determined the molecular basis of the interaction of MIA with the Hep II domain of fibronectin based on nuclear magnetic resonance spectroscopic binding assays. The authors have identified the type III modules 12 to 14 of fibronectin's Hep II as the major MIA binding sites. These results now provide a new target protein-protein binding interface for the discovery of novel antimetastatic agents against malignant melanoma in the future.

NMR-based Drug Development and Improvement Against Malignant Melanoma - Implications for the MIA Protein Family.

The Melanoma Inhibitory Activity (MIA) protein is strongly expressed and secreted by malignant melanoma cells and was shown to promote melanoma development and invasion. The MIA protein was the first extracellular protein shown to adopt an Src homology 3 (SH3) domain-like fold in solution that can bind to fibronectin type III domains. Together with MIA, the homologous proteins OTOR (or FDP), MIA-2, and TANGO (or MIA-3) constitute a protein family of non-cytosolic and - except for fulllength TANGO and TANGO1-like (TALI) - extracellular SH3-domain containing proteins. Members of this protein family modulate collagen maturation and export, cartilage development, cell attachment in the extracellular matrix, and melanoma metastasis. These proteins may thus serve as promising targets for drug development against malignant melanoma. For the last twenty years, NMR spectroscopy has become a powerful technique in medicinal chemistry. While traditional high throughput screenings only report on the activity or affinity of low molecular weight compounds, NMR spectroscopy does not only relate to the structure of those compounds with their activity, but it can also unravel structural information on the ligand binding site on the protein at atomic resolution. Based on the molecular details of the interaction between the ligand and its target protein, the binding affinities of initial fragment hits can be further improved more efficiently in order to generate lead structures that exhibit significant therapeutic effects. The NMR-based approach promises to greatly contribute to the quest for low molecular weight compounds that ultimately could yield drugs to treat skin-related diseases such as malignant melanoma more effectively.