The Expression, Purification, Structure And Function Of Ferric-ion Binding Protein And Its Mutant Y195F

Iron is an essential nutrient required for all living organisms. However, in humans and other mammals, iron tends to precipitate as ferric hydroxide at blood pH where the concentration of free Fe³⁺ is strictly controlled less than 10^-18 mol·L^-1. To maintain growth and multiplication, some pathogenic bacteria, such as Neisseria gonorrhoeae and N. meningitidis, have developed several efficient iron capture systems, including specific ferric-iron binding protein (FBP), to sequester iron from their hosts.The 34-kDa ferric-ion binding protein from Neisseria is a single chain protein, which contains 309 amino acids and folds into two domains. The structural analysis revealed that the Fe³⁺ binding sites, located in the"venus fly trap"motif between the two domains, consist of two Tyr, one His and one Glu residues, together with a phosphate (so called the synergistic anion) and a water molecule. Fe³⁺ binds to FBP reversibly, and can be removed from the protein (in this case, the protein is turned into its apo-form). Some other metal ions can also occupy the Fe³⁺ binding sites of the protein, which indicate that FBP could be the potential target for some antibacterial or anti-cancer metal ions.Protein phosphorylation is known as the most common and the most important process for the protein modification after the protein is translated. It participates in and even controls many life activities in vivo. Phosphorylation/dephosphorylation regulates and controls many cellular processes, such as signal transduction, gene expression, cell cycle and so on. Besides transporting Fe³⁺ across the periplasm, holo-FBP was also reported to display the phosphatase activity. However, the enzymic mechanism is unclear and apo-FBP has not been investigated so far.We reported here for the first time that apo-FBP can catalyse the hydrolysis of pyrophosphate (PPi). By using 31P NMR and UV-Vis spectroscopy, Western blotting assay, and ICP-AES analysis, the kinetics of the reaction between apo-FBP and PPi was investigated. The reaction followed zero-order kinetics, and is independent on the concentration of PPi, but is dependent on the concentration of the protein. The reaction rate constant was measured to be 1.02×10^-2, 1.04×10^-2, 0.10, and 0.135mmol·L^-1·h^-1 for [apo-FBP] = 36, 86, 107, 217μmol·L^-1, respectively, in 10mmol·L^-1 Hepes buffer at pH 7.40 and 293K. The reaction was also pH-dependent, with the rate constant at pH 8.5 being 1.2 times compared to pH 7.4, and being 1.18 times at pH 7.4 to pH 6.5, under the same condition (apo-FBP] = 75μmol·L^-1, [PPi]:[FBP] = 50:1, 10mmol·L^-1 Hepes buffer, 293K). Traces of Fe³⁺ in the solution can further accelerate the reaction. The rate constant with the presence of 2% Fe³⁺ was 1.6 times over that of apo-FBP. It was also found that holo-FBP catalysing the hydrolysis of PPi is 2.6 times faster than apo-FBP under the same condition.The FBP/Y195F mutant was expressed in E. coli and purified by chromatography. There was no bound Fe³⁺ in the mutant. The binding of Fe³⁺ to Y195F was much weaker than its wild type, and the binding constant is 1.4×10³L·mol^-1 for Y195F-Fe-(NTA)x (10mmol·L^-1 Hepes buffer, pH7.4, 298K). The rate constant of the PPi hydrolysis catalyzed by Y195F is only one tenth of that catalyzed by apo-FBP ([Y195F] = 79mmol·L^-1, [PPi]:[Y195F] = 50:1, in 10mmol·L^-1 Hepes buffer at pH 7.40 and 293K). Western blotting assay and ICP-AES analysis had shown that there was no tyrosine phosphorylation on apo-FBP, which is consistent with the absence of iron and phosphate in the protein. On the contrast, the tyrosine phosphorylation was observed on holo-FBP, the reaction product from apo-FBP with PPi, and that from holo-FBP with PPi, and each protein molecular contains about 1, 2 and 1 Pi, respectively.Based on the results above, we deduce that the mechanism of apo-FBP catalyzing PPi hydrolysis is through the tyrosine phosphorylation on the protein. The same mechanism is followed by holo-FBP after the Fe³⁺ was removed by PPi firstly. The phosphorylation site is Tyr195, the Fe³⁺ binding site of protein (whether Tyr196 is the phosphorylation site need to be further confirmed).A recombinant of Cu(Glu)₂-FBP was prepared by incubating apo-FBP with copper glutamate, which was to crystallize under different conditions. Crystals with proper size and regular appearance was harvested and analyzed by X-ray diffraction preliminarily. The crystal space group is C222, with the asymmetry unit parameters of nine protein molecules: a = 130.985A,b = 162.650A,c = 385.525?,α=β=γ= 90°.The fact, that FBP, iron transport protein of pathogens, showed the phosphatase activity to catalyze PPi hydrolysis, with the mechanism of phosphorylation on the Fe³⁺ binding site tyrosine(s), could have important significance in energy supply for the bacteria, and give a clue to design novel bacteriostatic agents targeted on this protein.