Structure-Function Investigation of Haemophilus Influenzae Ferric Binding Protein under Changing Environmental Conditions

Abstract

FbpA is an iron transport protein from the pathogenic bacteria, Haemophilus Influenzae, which hijacks one iron from human Transferrin (Tf) and transports it through the periplasmic space of the organism. It has 309 residues, and contains an iron binding cleft with two tyrosines, one histidine and one glutamic acid residues. Iron is held in the cleft by the octahedral coordination of these residues and a phosphate which serves as a synergistic anion. In this study, we investigated the significance of pH and ionic strength (IS) on the iron binding and release dynamics of H. influenzae ferric binding protein (FbpA) through binding/release assays as well as from a structural perspective. FbpA was expressed, purified and characterized in large quantities. Size Exclusion Chromatography (SEC), Dynamic Light Scattering (DLS), UV-Vis Absorption Spectroscopy (AS) and Small Angle X-ray Scattering (SAXS) were used for detecting structural changes. SAXS measurements were used to compare solution structures with the crystal structures, as well as to detect the changes in protein conformations under different environmental conditions. Additionally, absorbance values at 480 nm due to metal-to-ligand charge transfer caused by the interaction of tyrosine residues with the iron were monitored for investigation of iron binding dynamics under different IS conditions. Our results show that both apo and holo FbpA have flexible structures in solution with the former being slightly more open compared to the latter and both forms are more stable at high IS. Results are consistent with the closing of the iron binding pocket, when iron is captured, resulting in a decrease in hydrodynamic radius and radius of gyration. It is proposed that low IS can lead to instabilities in the protein structure and expose the bound iron to chelators at the inner membrane.