Characterization of a fold in TANGO1 evolved from SH3 domains for the export of bulky cargos.

Bulky cargos like procollagens, apolipoproteins, and mucins exceed the size of conventional COPII vesicles. During evolution a process emerged in metazoans, predominantly governed by the TANGO1 protein family, that organizes cargo at the exit sites of the endoplasmic reticulum and facilitates export by the formation of tunnel-like connections between the ER and Golgi. Hitherto, cargo-recognition appeared to be mediated by an SH3-like domain. Based on structural and dynamic data as well as interaction studies from NMR spectroscopy and microscale thermophoresis presented here, we show that the luminal cargo-recognition domain of TANGO1 adopts a new functional fold for which we suggest the term MOTH (MIA, Otoraplin, TALI/TANGO1 homology) domain. These MOTH domains, as well as an evolutionary intermediate found in invertebrates, constitute a distinct domain family that emerged from SH3 domains and acquired the ability to bind collagen.

Deciphering the Emulsification Process to Create an Albumin-Perfluorocarbon-(o/w) Nanoemulsion with High Shelf Life and Bioresistivity.

This work aimed at the development of a stable albumin-perfluorocarbon (o/w) emulsion as an artificial oxygen carrier suitable for clinical application. So far, albumin-perfluorocarbon-(o/w) emulsions have been successfully applied in preclinical trials. Cross-linking a variety of different physical and chemical methods for the characterization of an albumin-perfluorocarbon (PFC)-(o/w) emulsion was necessary to gain a deep understanding of its specific emulsification processes during high-pressure homogenization. High-pressure homogenization is simple but incorporates complex physical reactions, with many factors influencing the formation of PFC droplets and their coating. This work describes and interprets the impact of albumin concentration, homogenization pressure, and repeated microfluidizer passages on PFC-droplet formation; its influence on storage stability; and the overcoming of obstacles in preparing stable nanoemulsions. The applied methods comprise dynamic light scattering, static light scattering, cryo- and non-cryo-scanning and transmission electron microscopies, nuclear magnetic resonance spectroscopy, light microscopy, amperometric oxygen measurements, and biochemical methods. The use of this wide range of methods provided a sufficiently comprehensive picture of this polydisperse emulsion. Optimization of PFC-droplet formation by means of temperature and pressure gradients results in an emulsion with improved storage stability (tested up to 5 months) that possibly qualifies for clinical applications. Adaptations in the manufacturing process strikingly changed the physical properties of the emulsion but did not affect its oxygen capacity.

Structural insights into photosystem II assembly.

Biogenesis of photosystem II (PSII), nature's water-splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate from Thermosynechococcus elongatus at 2.94 Å resolution. It contains three assembly factors (Psb27, Psb28 and Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace the bicarbonate ligand of non-haem iron with glutamate, a structural motif found in reaction centres of non-oxygenic photosynthetic bacteria. These results reveal mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water-splitting reaction.

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.

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.

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.