BackgroundSmall G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown.ResultsBased on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7).ConclusionOur analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

A combination of biophysical techniques has been used to characterize the interaction of an antitrypanosomal agent, CGP 40215A, with DNA. The results from a broad array of methods (DNase I footprinting, surface plasmon resonance, X-ray crystallography, and molecular dynamics) indicate that this compound binds to the minor groove of AT DNA sequences. Despite its unusual linear shape that is not complementary to that of the DNA groove, a high binding affinity was observed in comparison with other similar but more curved diamidine compounds. The amidine groups at both ends of the ligand and the -NH groups on the linker are involved in extensive and dynamic H-bonds to the DNA bases. Complementary and consistent results were obtained from both the X-ray and molecular dynamics studies; both of these methods reveal direct and water-mediated H-bonds between the ligand and the DNA.