Importance of the alpha-I helix domain of IFABP in the fatty acid transfer to phospholipid membranes.

GISELA FRANCHINI, LISANDRO FALOMIR, BETINA CORSICO Y JUDITH STORCH.

Waterville Valley, New Hampshire, USA.

Congreso; Molecular and cell biology of lipids Gordon Research Conferences; 2005

Intestinal FABP (IFABP) belongs to a family of small cytosolic proteins involved in lipid transport and metabolism that share a common structure. IFABP structure consists of ten antiparallel â-strands that form a â-barrel, which is capped by two short á-helices arranged as a helix-turn-helix segment. It is believed that this helical domain is part of a dynamic portal that regulates fatty acid (FA) entry and exit from the internal binding cavity. Previously, we demonstrated employing an energy transfer assay (FRET) that FA transfer from IFABP occurs during protein-membrane collisional interactions, in which ionic interactions between positively charged residues on the protein surface and negatively charged phospholipid headgroups are involved. Contrarily, other members of this family show an aqueous-diffusional ligand transport mechanism to acceptor membranes, like Liver FABP (LFABP). We have also shown, using a helix-less variant of IFABP (IFABP-HL) that the á-helical region of IFABP is involved in membrane interactions, and appears to play a primary role in the collisional mechanism of fatty acid transfer from IFABP to membranes. In the present study, the role of specific lysine residues of the portal region of IFABP was directly examined using site-directed mutagenesis. We have engineered a series of point mutant proteins of IFABP where lysine’s positive charge of the á-helical domain were eliminated and reverted. We also included a pair of mutants of the â-barrel as controls. All mutants were studied for conformational and binding site integrity and showed non relevant modifications compared to the wild type. Employing a fluorescence resonance energy transfer assay we analyzed the rates and mechanism of FA transfer to phospholipid membranes. Most of the á-helical domain mutants showed slower rates of FA transfer to zwitterionic small unilamellar vesicles (SUVs), but only a significant modification of the absolute rate was observed for mutant K27I of the á-II helix; which also showed a drastic modification of the FA transfer mechanism to aqueous-diffusional. Sensitivity to negatively charged SUVs was also reduced; the mutants with charge reversion in the á-II helix were the most affected. These results demonstrate that the lysines of the á-helical region of IFABP participate of the protein-membrane collisional mechanism of fatty acid transfer to membranes. Furthermore, it appears that the á-II segment is more important in the charge-charge interaction. On the other hand, crosslinking experiments with the lipid photoactivable reagent 125I-TID-PC showed physical interaction of wild-type IFABP with phospholipid vesicles, which was modulated by membrane’s composition and the presence of ligand. All point mutants showed similar ability to interact with membranes compared to the wild type protein. We also constructed a chimeric protein, in which the á-I helix was exchanged between IFABP and LFABP, to obtain a protein with the binding pocket of IFABP and á-I helix of LFABP. This protein seems to interact less than the wild type protein. Only if the helical domain is completely removed, as in the IFABP-HL, the protein loses the ability to interact with membranes. These results showed that the á-helix region is important in protein-membrane interaction, particularly the á-I helix of IFABP, which happens to be an anphipatic helix. The analysis of FA transfer mechanism and protein-membrane interaction performed in this work allowed us to elucidate the role of different domains and single residues that can now be recognized as structure determinants for these functions.