Identification and structural characterization of a Fatty Acid-Binding Protein from Leishmania amazonensis

Leishmania parasites rely on host-derived lipids and fatty acids for energy metabolism, differentiation, immune modulation, and drug susceptibility, yet the mechanisms underlying lipid acquisition and utilization remain poorly understood. Members of the calycin superfamily, which includes lipocalins and fatty acid-binding proteins (FABPs), are central to intracellular lipid transport in other organisms. Analysis of Leishmania genomes identified a conserved gene, lfabp1, encoding a hypothetical modular protein comprising an N-terminal region with two lipocalin motifs and a C-terminal FABP-like domain (Lei-FABP). Phylogenetic analyses revealed strong conservation of lfabp1 across Leishmania species and probable orthologs in Trypanosoma cruzi. Furthermore, the lipocalin–FABP domain architecture appears to be restricted to kinetoplastids. To characterize the structural properties of LFABP1 C-terminal domain (Lei-FABP), it was heterologously expressed in Escherichia coli, purified, and crystallized. X-ray diffraction data up to 1.8 Å were collected at the Manacá beamline (Sirius/CNPEM). The structure, calculated by molecular replacement, revealed a canonical FABP fold: a closed β-barrel capped by a helix–loop–helix motif, consistent with its lipid-binding function. A Lei-FABP sample was delipidated using a Lipidex-1000 resin, and changes in the methyl region of the ¹H NMR spectrum were observed, suggesting removal of possible hydrophobic ligands. NMR titration assays demonstrated preferential interaction with dodecanoic acid (C12:0), a result further corroborated by tryptophan-fluorescence assays. Finally, the full-length LFABP1 was detected in promastigote and amastigote forms of L. amazonensis by western blot. Ongoing characterization of CRISPR-Cas9-generated knockout and add-back strains should clarify its functional role in lipid metabolism and parasite pathogenicity.