Abstract: In budding yeast Sic1, an inhibitor of cyclin-dependent kinase (Cki), blocks the activity of Cdk1-Clb5/6 (S-Cdk1) kinase required for the initiation of DNA replication that takes place only when Sic1 is removed . Deletion of Sic1 causes premature DNA replication from fewer origins, extension of the S-phase and inefficient separation of sister chromatids during anaphase, whereas delaying S-Cdk1 activation rescues both S and M phase defects. Despite the well documented relevance of Sic1 inhibition on S-Cdk1 for cell cycle control and genome instability, the mechanism by which Sic1 inhibits S-Cdk1 activity remains obscure. Sic1 has been proposed to be a functional homologous of mammalian Cki p21Cip1, that is characterized by a significant sequence similarity with Cki p27Kip1, inhibitor of the Cdk2/Cyclin A kinase activity during S-phase.

Abstract: The information carried by the aminoacidic sequence can be used by different bioinformatic methods, in order to predict the 3D structure of a protein or its domains. The tools for sequence alignment permit to individuate homologous regions among proteins, and this represent the basis for a homology modeling procedure. The algorithms of secondary structure prediction use chemical, physical and statistical parameters to recognize if a region of sequence could assume a specific secondary structure. Fold recognition servers can test if a protein sequence is compatible with one of the known folds in the PDB. If these different tools give rise to homogeneous responses, it is possible to predict with good reliability the fold of a protein or single domains of unknown structure. hSos1 is a multidomain protein involved in the activation of the Ras signaling by catalyzing guanine nucleotide exchange on Ras. The Ras-GEF domain of hSos1 (Sos-Cat) is flanked by amino- and carboxyl-terminal regions, which are able to inhibit hSos1 activity towards Ras. To investigate the structural determinants of this inhibition, it is necessary to know the structural features of the involved domains. The carboxyl terminus of hSos1 contains a proline rich domain with consensus sequences for binding to the SH3 domains, while the amino-terminal region of hSos1 includes three domains: Histone domain, Dbl Homology domain (DH) and Pleckstrin homology domain (PH). The Histone domain is involved in the inhibition of the Ras-GEF activity of hSos1. It can also bind the PH domain, while it cannot interact with the DH domain. The DH domain is implicated in the inhibition of the Ras-GEF activity of hSos1, possibly through direct interaction with Sos-Cat. The PH domain is able to interact with the DH domain; the crystal structure of the PH-DH complex is available. We have focused on the intra-molecular interactions that occur among these domains in the activation/inhibition of hSos1 by means of computational tools, like the low-resolution protein-protein docking. The essence of the procedure is the reduction of protein structures to digitized images on a three-dimensional grid. The structural elements smaller than the step of the grid (e.g., atom-size) are not present in the docking. This feature permits to reduce the negative effect of structural changes upon complex formation on docking calculation.

Abstract: Histone folds are structural elements that are able to form dimers by means of tight interactions between hydrophobic surfaces. Normally, a histone fold is composed by a long alpha-helix flanked by two or three shorter helices. In the nucleosome core particle, two pairs of H2a-H2b and H3-H4 histone heterodimers assemble together, giving rise to a disk-like octamer upon which DNA rolls up. The publication of the X-ray structure of the prokaryotic histone from Methanopyrus kandleri highlighted a novel protein fold, which is originated by the assembly of two consecutive histone folds included in the same peptide chain. More recently, the publication of the X-ray structure of the amino-terminal domain of hSos1 showed that also this protein module assumes a similar fold, dubbed the histone pseudodimer and here also referred to as “double histone fold”. In fact, the evolutionary relationship between the H2a histone and the domain spanning the protein sequence 96-190 of hSos1 had been already disclosed, due to high sequence similarity between the two domains. However, the first histone-like domain of hSos1, spanning the protein portion 6-95, does not show evident sequence similarity with histones (Sondermann et al, 2003). Moreover, it is unknown whether the double histone fold can be found in other protein families. In view of this, we initiated an in silico study aiming at the identification of other proteins characterized by a sequence that is compatible with the double histone fold.

Abstract: The analysis of protein 3D structure is an important step to understand their mechanism of action, regulation, function and family’s belonging. Experimental methods for proteins structure determination don’t keep up with the increasing number of genomic sequence available: this led to an increase of computational methods that predict three-dimensional model for a protein of unknown structure (target) on the basis of sequence similarity to proteins of known structure (templates). There are different kinds of Homology Modelling methods, but all of them can’t recover from an incorrect target-template alignment: a good alignment is the first think to be considered when we’re talking about model’s confidence. SWISS MODEL, an automated comparative protein modelling server starts with the analysis of the structural conserved regions in the target-templates alignment. Ras protein are highly conserved GTPase playing a pivotal role in different important cellular events: cell proliferation, differentiation, cellular traffic and cytoskeleton organization. Within cells, Ras proteins exist both in a GTP-bound form (“on” state) or a GDP-bound (“off” state). The level of the GTP-bound state derives from the balance of the activity of the GTPase Activating Proteins (GAPs) and Guanine nucleotide Exchange Factors (GEFs). Common feature of all Ras GEFs is the presence of a domain, the RasGEF domain, carrying all the main structural features needed to interact with Ras and to exchange the nucleotide. A notable feature of this catalytic domain is the protrusion of a hairpin, formed by helices αH and αI, out of the core of the domain. It has been proposed helix αH plays an important role in the nucleotide-exchange mechanism opening up the nucleotide-binding site.

Abstract: Ras proteins are small GTPases ivolved in signaling pathways controlling cell growth and differentiation. They act as molecular switches by cycling between an active GTP- and an inactive GDP-bound state. Following the activation of specific cell-surface receptors, Ras proteins switch from inactive to active state through the catalytic action of specific Guanine nucleotide Exchange Factors (GEFs), that promote the dissociation of GDP from Ras, allowing GTP entrance into the Ras nucleotide poket. The Saccharomyces cerevisiae Ras-GEF Cdc25 (Cdc25Sc) was the first Ras-exchanger to be identified. In higher eukaryotes there are two different classes of Ras-specific Cdc25Sc homologs, Sos proteins and Cdc25Mm, also referred to as Ras GRF. Ras-specific GEFs are made of several functional and structural domains, Ras GEF activity is contained within a domain showing very high similarity to the Cdc25Sc catalytic domain and called, for this reason, Cdc25 homology domain. Structural studies on Ras crystallized in complex with nucleotide (GDP or GTP-analogs) and human exchange factor Sos respectively have allowed both to identify conformational differences between active and inactive state of Ras, and to make hypothesis on molecular determinants of interaction and catalytic activity of human Sos. Mutational and structural studies on Ras GEFs catalytic domain have pointed to a major role for the helical-hairpin formed by αH and αI helixes (catalytical hairpin) in the catalytic mechanism of Ras-specific GEFs. In the present work we investigate the Ras GEF αG-helix role in Ras-GDP to GTP exchange.