High resolution structure of an alternate form of the ferric ion binding protein from Haemophilus influenzae

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Pathogenic Gram-negative bacteria require iron for survival.
• Periplasmic iron binding proteins (FbpA) are crucial for acquiring iron from external sources.
• The wild-type Haemophilus influenzae FbpA binds iron using specific amino acids and a phosphate ligand.

Purpose of the Study:

• To determine the high-resolution crystal structure of an iron-loaded mutant periplasmic ferric ion binding protein (FbpA) from Haemophilus influenzae.
• To investigate the iron-binding properties of the H9Q FbpA mutant.
• To explore potential alternative iron-binding mechanisms of FbpA.

Main Methods:

• Site-directed mutagenesis to create the H9Q FbpA mutant.
• Expression and purification of the FbpA mutant in Escherichia coli.
• Iron loading of the purified protein.
• High-resolution (1.14 Å) X-ray crystallography to determine the protein structure.

Main Results:

• The crystal structure of the iron-loaded H9Q FbpA mutant was determined.
• The H9Q mutant binds ferric iron in an open conformation.
• Unexpectedly, EDTA was found bound to the H9Q FbpA mutant at the anion binding site, coordinating iron along with Tyr195 and Tyr196.

Conclusions:

• This is the first reported structure of a metal-binding protein complexed with EDTA.
• The results suggest that FbpA may be capable of binding and transporting iron complexed with biological chelators, in addition to free ferric iron.
• This broadens the understanding of iron acquisition strategies in pathogenic bacteria.