News and Publications

Crystallography of Iron Metalloproteins

D.E. McRee, C. Bruns, P. Williams, V. Shridhar, N. Jourdan, C. Shipke, R. Nunn

Our laboratory studies the structure, function, and catalysis of metalloproteins, with particular emphasis on iron metalloproteins. One question being investigated is how protein can control iron and give it diverse biological roles ranging from electron transfer to oxygen activation to iron transport. We are using high-resolution protein crystallography coupled with protein engineering and biochemistry to answer these questions.

IRON-BINDING PROTEIN

Iron is an essential growth requirement of all organisms and using lactoferrin and transferrin to sequester iron from invading pathogens is one means of antibacterial defense in humans. However, some bacterial pathogens, notably Neisseria and Haemophilus, have evolved to turn adversity into advantage; they have receptors for transferrins that enable the bacteria to steal the host iron. This iron is transported into the pathogens by a bacterial protein, iron-binding protein. We solved the structure of the iron-binding protein to high resolution and have started protein engineering studies. Our goal is to find a way to knock out this protein in bacteria and thus produce a bacteriostatic agent.

CYTOCHROMES

Cytochrome c552 is the physiologic partner of cytochrome c oxidase in Thermus thermophilus. We are solving the structure of cytochrome c552 in collaboration with E. Stura, Department of Molecular Biology, and J. Fee, University of California, San Diego. We also have crystals of the CuA fragment of T thermophilus cytochrome c oxidase (Fig. 1), the electron transfer partner of c552. The combination of the two gives us a unique opportunity to study an electron-transfer system. We are doing protein engineering studies in collaboration with J. Fee.

Although more than 600 mammalian P-450 isozymes exist, the structures of these membrane-bound P-450s have not been determined. In collaboration with E. Johnson, Department of Molecular and Experimental Medicine, we have crystallized the first diffraction-quality crystals of a rabbit liver P-450. We have preliminary multiwavelength anomalous diffraction data and should have a structure soon.

VERY HIGH-RESOLUTION METAL-SITE STRUCTURES

With the advent of freezing devices and synchrotron radiation sources, very high-resolution structures (<1.4 Å) are now routinely accessible. At these resolutions, it becomes possible to refine structures with the rigorous methods used in small-molecule crystallography. This refinement includes adding hydrogens and anisotropic thermal factors and using full-matrix least-squares analyses to determine the standard uncertainties of the atom positions. Because metal centers scatter more than the lighter carbon atoms do, the metal centers are even better determined.

In a high-resolution 1.35-Å refinement of Azotobacter 7-iron ferredoxin, we determined the positions of the iron and sulfur atoms with a standard uncertainty of 0.01 Å. This uncertainty is within the limit of molecular orbital calculations and will lead to a better coupling of theoretical calculations on metal centers and structure. We also refined cytochrome c peroxidase to 1.38-Å resolution (Fig. 2). These more accurate coordinates will allow improved calculations of the enzyme's mechanism. We plan to build up a database of high-resolution metalloprotein structures in conjunction with the Scripps Metalloprotein Structure and Design Group (http://www.scripps.edu/pub/dem-web/metallo/), of which our group is a member.

COMPUTATIONAL CRYSTALLOGRAPHY

As part of a project funded by the National Science Foundation to improve visualization and computational tools for protein crystallography, we developed a software package called XtalView (http://www.scripps.edu/pub/dem-web/toc.html). XtalView uses the power of modern workstations to solve protein crystallographic problems by using a visual, graphical user interface. XtalView is available from the Computational Center for Macromolecular Structure (http://www.sdsc.edu/CCMS/) and has been downloaded by more than 1000 groups.

PUBLICATIONS

Bruns, C.M., Nowalk, A.J., Arvai, A.S., McTigue, M.A., Vaughan, K.G., Mietzner, T.A. McRee, D.E. Structure of Haemophilus influenzae Fe³⁺-binding protein reveals convergent evolution within a superfamily. Nature Struct. Biol. 4:919, 1997.

Cao, Y., Musah, R.A., Wilcox, S.K., Goodin, D.B., McRee, D.E. Protein conformer selection observed by protein crystallography. Protein Sci. 7:72, 1997.

McRee, D.E. A brief history of protein crystallographic computing. Rigaku J. 15:1, 1998.

McRee, D.E. Living with metal ions. Nature Struct. Biol. 5:8, 1998.

McRee, D.E., Israel, M. XtalView. In: Crystallographic Computing 7. Bourne, P., Watenpaugh, K. (Eds.). Oxford Press, New York, in press. Available at: http://www.sdsc.edu/projects/Xtal/IUCr/CC/School96/IUCr.html.

Musah, R.A., Jensen, G.M., Rosenfeld, R.J., Bunte, S.W., McRee, D.E., Goodin, D.B. Variation in strength of a CH to O hydrogen bond in an artificial cavity. J. Am. Chem. Soc. 119:9083, 1997.

Stout, C.D., Stura, E., McRee, D.E. The 1.35-Å resolution structure of 7-Fe ferredoxin and the geometry of the Fe-S clusters with bond standard uncertainties of 0.01 Å. J. Mol. Biol. 278:629, 1998.