Abstract: Phosphatidylinositol kinases are found in all eukaryotes and serve important functions in phosphatidyl-inositol (PI) signaling pathways. Recently, a new subfamily of the PI kinase superfamily involved in meiotic and V(D)J recombination, chromosome maintenance and repair, cell cycle progression and cell cycle checkpoint has emerged, called PIK-related. This family includes ATM, ATR, DNA-PK, ESR1, Rad3, TOR1, TOR2, FRAP, TEL1 kinases. These are large proteins (2000-4000 aa) that only share similarity in the ~300 aa kinase domain to classical PI kinases. Another group distantly related to PI kinases comprises the TRRAP proteins. They also share similarity to the PI kinase domain however they lack the catalytic residues and indeed none of them has been shown to possess kinase activity. It has previously been noted that the TRRAP and PIK-related proteins share a unique motif at the C terminus. Analysis of the remaining sequence has so far not been able to clearly define shared domains in the large N-terminal portions. We here describe a novel homology domain spanning ~500 aa, N-terminal to the PI kinase domain in the PIK-related and TRRAP subfamilies. We call this domain FAT after representatives of the three main groups sharing the domain (FRAP, ATM, and TRRAP). This domain is only present in the FRAP, ATM and TRRAP subfamilies, it is not found outside these subfamilies and always coexists with the C terminal domain previously identified. It is possible that they fold together in a configuration that is necessary for proper function of the PI kinase domain, which is wedged in between the FAT and the C-terminal domains.

Abstract: Trop-1 and Trop-2 are homologous transmembrane glycoproteins that are expressed at high levels by most human cancer cells. Trop-1 is induced by cell proliferation and oncogenic transformation, and is a marker of epithelial progenitor cells. Trop-2 is expressed by terminally differentiated epithelial cells and by the placental trophoblast. In the N-terminal region of both molecules there is a large cysteine-rich region (98 residues in Trop-1 and 111 in Trop-2) that is conserved in all the Trop molecules cloned so far. The last six cysteines of this region constitute a typical thyroglobulin domain, while the first six cysteines do not conform to any typical PROSITE pattern. Sequence analysis, secondary structure prediction, fold recognition indicate that an EGF-like domain can be recognized in the region involving the first six cysteines of the Trop molecules. Moreover, the results of sequence analysis show that this sequence module formed by an EGF domain and a thyroglobulin domain is also present in other proteins involved in cell adhesion and in growth control, and hence a role of such a module in these functions can be proposed. The Swiss-PDB-Viewer homology modelling program was employed to build a 3D model of the thyroglobulin domain of the Trop molecules. As a template, we used the thyroglobulin domain of the p41 protein. The RMSD between target and template structures is 1.36 Å. Moreover, 87% of the predicted 3D structure residues fall in the core region of the Ramachandran Plot and 100% in its acceptable region. We are working on the prediction of the 3D structure of the EGF region: joining the two models, a final prediction of the complete cysteine-rich region of the Trop molecules could be obtained.

Abstract: In order to investigate structural/functional relationships of a wheat subtilisin-chymotrypsin inhibitor (WSCI) (1), we decided to explore its secondary and tertiary structures by applying an homology modelling procedure (using the Modeller program as part of the Quanta package) (2). The barley chymotrypsin inhibitor CI-2A (3), which exhibits 89% sequence similarity with the wheat inhibitor, has been chosen as reference structure. The best model structure obtained for WSCI, shows that 50% of its amino acid sequence (72 residues) are involved in motifs of secondary structure; particularly, 11 amino acid residues give rise to an helix, 12 residues form a long loop connecting two parallel strands, each made up of 6 residues. In the spatial model, the relative positions of such motifs are in agreement with the experimental data obtained upon interaction between WSCI and subtilisin; in fact, the bacterial proteinase cleaves, specifically, the inhibitor peptide bond Met48-Glu49 located in the middle of the above connecting loop. The weak interactions observed (H-bonds and salt bridges) in WSCI model are in perfect agreement with those found in the reference structure of CI-2A. Investigations regarding the modality of interaction between WSCI and susceptible proteinases are in progress.

Abstract: The rhodopsin family of receptors employs guanine nucleotide binding proteins (G proteins) to transduce signals across the cell membrane. All the G protein coupled receptors (GPCRs) share the presence of seven hydrophobic regions that are believed to form a bundle of a-helical transmembrane domains connected by alternating intracellular and extracellular hydrophilic loops. Three-dimensional model building and molecular dynamics (MD) simulations of the a1b-adrenergic receptor (a1b-AR), of the oxytocin receptor (OTR) and of the luteinizing hormone receptor (LHR) were employed to provide hypotheses about the molecular mechanisms underlying the mutation-induced activation of these GPCRs. The comparative analysis of the wild type receptors and of several constitutively active or inactive mutants was instrumental to infer the structural/dynamics features which could characterize the active and the inactive forms of these receptors. These features were also employed for predicting the functional behavior of new receptor mutants. Rigid body docking simulations between the functionally different forms of the a1b-AR and the LHR, on one hand, and heterotrimeric G proteins, on the other, suggested that the cytosolic crevice shared by the constitutively active receptor structures and formed by the second and the third intracellular loops as well as by the cytosolic extensions of helices 3, 5 and 6, might participate to receptor-G protein interface. The results of this study might provide a structural framework to interpret the pathological effects induced by naturally occurring mutations of the LHR. In addition, the theoretical models here proposed can be useful for designing new mutations or ligands able to modulate receptor function as well as to drive experiments aimed at exploring the receptor-G protein interface.