Structural and functional characterization of IivA protein of Brucella abortus

CARRICA M; CRAIG PO; ALONSO S; GOLBAUM FA; SABIO Y GARCIA J; ROSSETTI O; CRAVERO S

Montevideo, Uruguay

Congreso; 6th International Conference of Biological Physics (ICBP), 5th Southern Cone Biophysics Congress and Biophysical Society of Argentina (SAB) 36th Annual Meeting; 2007

IivA is a 113-residue basic protein that is involved in Brucella abortus virulence in mice. Although IivA protein is highly conserved across diverse bacterial species, its structure and function are unknown. The primary sequence analysis of IivA predicts a highly a-helix content and two coiled-coil regions encompassing the C- and N-terminal halves of the molecule. By chemical cross-linking and static light scattering experiment of complete IivA and truncated forms of the N and C terminal coiled-coil regions (IivA-N and IivA-C respectively), we demonstrated that Iiva self-associates as a trimer through its C-terminal half.  We carried out circular dichroism and limited proteolysis experiments of IivA, IivA-N and IivA-C and found that while the C-terminal is mainly a-helix and resistant to proteolysis, the N-terminal is natively unfolded with propensity to form an amphipathic a-helix. This amphipathic helix can be formed in the presence of moderate concentration of phosphate, sulphate and chloride ions, and also in the presence of SDS micelles and low pH. These results and the occurrence of trimeric coiled coil in several fusogenic proteins prompted us to investigate the interaction with phospholipids vesicles. We measured the CD spectrum of IivA in the presence of vesicle composed of different ratios of phosphatidic acid (PA) and phosphatidylcholine (PC). In all conditions, we observed a significant increase in the a-helical content of IivA and IivA-N. The maximum effect was produced by vesicles composed of 25% PA and 75% PC. To study IivA association to the vesicles, we constructed two single-Trp mutants (IivA-L14W and IivA-S114W), that span the N- and C-terminal domains, respectively. We found that the fluorescence maximun of IivA-L14W shifts to lower wavelength upon mixing with vesicles, while the IivA-S114W mutant showed no shift. This suggests that the Trp in the N-terminal region is relocated into a hydrophobic core. Furthermore, we investigated the fusogenic activity of IivA by measuring the size increment of vesicles after addition of protein using Dynamic Light Sacttering, and the lipid membrane mixing by FRET. These experiments, demonstrated that IivA has an in vitro membrane-fusion activity that requires the full length protein, as the isolated carboxyl and amino terminal domains have none or a moderate activity.