Bacterial Fe(III)-binding protein (FBP)

I determined the atomic structure of this protein by X-ray crystallography using the technique of multiple isomormphous replacement (MIR).

Fe(III)-binding protein is used by pathogenic bacteria to absorb iron from the host environment. The opportunistic pathogenic bacterium Haemophilus influenzae is the most common cause of bacterial meningitis in children aged six months to five years, and is responsible for many respiratory tract infections such as pneumonia, bronchitis, sinusitis, and oditis media. This pathogen relies upon siderophore-independent iron acquisition from host proteins for its survival. Vertebrate animals sequester iron within the proteins transferrin and lactoferrin, thereby providing a non-specific bacteriostatic extracellular environment. Haemophilus overcomes this hostile environment by specifically utilizing lactoferring and transferrin as iron sources, shuttling iron from lactoferrin/transferrin receptors on the bacterial outer membrane to the inner membrane/cytoplasm via a 37 kD periplasmic ferric iron-binding protein (FBP). FBP binds iron with an extremely high affinity of 10^19 M^-1.

This work is being done in collaboration with Tim Mietzner. My mentor for this project is Duncan McRee.

Crystals

These crystals are grown by vapor diffusion using the hanging drop method. Two of the crystal forms shown were discovered by Andy Arvai, the other by myself.

Atomic coordinates

The following link retrieves the atomic coordinates from the Brookhaven Protein Data Bank.
Download the atomic coordinates of Haemophilus FBP
If you use these coordinates, please cite:

C.M. Bruns, A.J. Nowalk, A.S. Arvai, M.A. McTigue, K.G. Vaughan, T.A. Mietzner, and D.E. McRee (1997) Structure of Haemophilus influenzae Fe+3-binding protein reveals convergent evolution within a superfamily. Nature Structural Biology 4(11): 919-924.

MIR Phasing

After years of effort screening hundreds of heavy atom conditions, no derivatives had been identified. A cysteine-containing mutant was generated which provided a mercury derivative that was then used to identify previously unrecognized derivative data sets. The cysteine mutation was chosen using the crystal structure of maltodextrin-binding protein, and a short stretch of apparent sequence conservation with FBP. This strategy worked perfectly, providing a mercury binding site on a concave surface region of the protein on the first try. FBP shares only 18% sequence identity with maltodextrin-binding protein, and all of a vast array of molecular replacement strategies were unsuccessful.

Atomic structures

Ribbon representation of the atomic structure of FBP from Haemophilus influenzae. The iron atom is the red sphere in the center.

1.6 Å electron density map of the active center of FBP from Haemophilus influenzae. The map is contoured at 2 and 9 standard deviations.

Literature References

T.A. Mietzner, S.B. Tencza, P. Adhikari, K.G. Vaughan, and A.J. Nowalk (1998) Fe(III) periplasm-to-cytosol transporters of gram-negative pathogens. Curr Top Microbiol Immunol 225: 113-135

"News and Views" companion piece to above:
E.N. Baker (1997) Iron-ic twists of fate. Nature Structural Biology 4(11): 869-871.

C.M. Bruns, A.J. Nowalk, A.S. Arvai, M. A.McTigue, T.A. Mietzner, and D.E. McRee (1997) Crystal Structure of Haemophilus Ferric-binding Protein: Insights into the Evolution of the Transferrin Superfamily. Presented at the West Coast Protein Crystallography Workshop, Asilomar conference center, Asilomar, California. March 15-19, 1997

C.M. Bruns, A.S. Arvai, A.J. Nowalk, T. A. Mietzner, D.E. McRee, and J.A. Tainer (1996) Structure Analysis of Key Drug Design Target Enzymes from Human Pathogens. Presented at the meeing of the International Union of Crystallography, Seattle, Washington, August 8-17, 1996.

Adhikari, P.A., Kirby, S., Nowalk, A.J., Veraldi, K.L., Schryvers, A.B., and Mietzner, T.A. (1995) Biochemical characterization of a Haemophilus influenzae periplasmic iron transport operon. J. Biol. Chem. 270:24719-24726.

Nowalk, A.J., Tencza, S.B., and Mietzner, T.A. (1994) Coordination of iron by the ferric iron binding protein (Fbp) of pathogenic Neisseria is homologous to the transferrins. Biochemistry 33:12769-12775.

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