The McRee Group, Molecular Biology, The Scripps Research Institute

last updated Nov. 11, 1999

Projects:

Fe-binding Protein
High-resolution metalloprotein refinement and error analysis
Cytochrome c Peroxidase Engineering
CuA center of cytochrome c oxidase
Thermus thermophilus Cytochrome c552
Microsomal Cytochrome P-450
Cyclic Peptide Nanotubes
Novel MAD Phasing Methods
XtalView Software

Pathogenic bacteria have developed unique mecahnisms for stealing iron from their hosts.  We have solved the structure of one of a protein, Fe-binding protein from Neisseria ghonorhea and Haemophilus influenzae and in collaboration with Dr. Tim Mietzner at the University of Pittsburgh and in collaboration with Dr. Anthony Schryvers at the University of Calgary, that is used to tranposrt iron into the bacteria.  We are using protein engineering to study the iron-binding mechanism ith the long-range goal of developing new antibacterial agents and understanding the molecular mechanism of iron transport. See Bruns et. al., Nature Struct. Biol., 4, pg. 919 and News and Views pg. 869 same issue.

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

We are using ultra-high resolution data and full matrix error analysis with SHELX-97 to build a database of accurate metalloproteins in different oxidation and spin-states.  We are pioneering methods for determining the error in metal-ligand bonds at unprecedented accuracy in order to determine the subtle changes of metal-ligand geometry upon redox changes and the extent to which proteins can distort and thus control metal site chemistry.

Stout, C.D., Stura, E.A., McRee, D.E. (1998)  "1.35 Å Structure of Azotobacter 7-Fe Ferredoxin and Determination of Fe-S Cluster Positions to 0.01Å Precision."  J. Mol. Biol. 278, 629-639.

Cytochrome c peroxidase protein engineering studies in collaboration with Dr. Goodin's lab at Scripps.  Recent publications include:

Wilcox, S.K., Putnam, C.D., Sastry, M., Blankenship, J., Chazin, W. J., McRee, D.E. and Goodin, D. B. (1998) "Rational Design of a Functional Metalloenzyme: Introduction of a Site for Manganese Binding and Oxidation into a Heme Peroxidase."  Biochemistry 37, 16853-16862.

Cao, Y., Musah, R.A., Wilcox, S.A., Goodin, D.B. and McRee, D.E.  (1998)  "Protein Conformer selection by Ligand Binding Observed with Protein Crystallography."  Protein Science 7, 72-78.

Fitzgerald, M.M., Musah, R.A., McRee, D.E. and Goodin, D.B. (1996)  "A Ligand-Gated Hinged Loop Rearrangement Opens a Channel to a Buried Artificial Cavity."  Nature Structural Biology 3, 626-631.

Cytochrome oxidase is the terminal step in the electron transport chain of oxidative phosphorylation.  Until recently and despite many years of research, the precise details of the geometry of the CuA center were not well-known.  Using an engineered fragment of the CuA subunit from Thermus thermophilus CuA, we have solved a crystal structure to very high resolution and determined the geometry with a high degree of precision, in collaboration Dr Jim Fee at UCSD.  We now know, for instance that the Cu-Cu distance is 2.51Å +/- 0.02Å.  Using these results, other groups are now re-examining the electron tranport and electronic properties of the CuA center.  We are now beginning mutagenesis studies.

Williams, P.A. Blackburn, N. J., Sanders, D., Bellamy, H., Stura. E.A., Fee, J.A. & McRee, D.E.. (1999) "The CuA Domain of thermus thermophilus ba3-type cytochrome c oxidase."  Nature Structural Biology, Vol. 6, 509-516..

Cytochrome c552, is the electron transport partner of T. thermophilus cytochrome oxidase . We are studying how this protein docks with the CuA subunit and rapidly transfers electrons into cytochrome c oxidase in the last step of the oxidative phosphorylation chain.
 
 

P450s are one of the most and numerous enzymes in the human body responsible for a diverse number of reactions.  Yet, no crystal structure of a mammalian P450 exists, although weveral bacterial P450s have been solved.  Using material engineered and expressed in the lab of Eric Johnson, we have suceeded in crystallizing the P450 CYP2C5, progesterone 21-hydroxylase, and solved its structure using MAD phasing.
 
 

We also solve nanotube structures in conjuction with Dr. Reza Ghadiri here at Scripps. Nanotubes are made up of cyclic peptides that self-assemble into infinite tubular structures of 9-12 Ångstroms diameter.

Ghadiri, M.R., Granja, J.R., Milligan, R.A., McRee, D.E. and Khazonovich, N. (1993) "Self-assembling Organic Nanotubes."  Nature366, 324-27.
Clark, T.D., Buriak, J.M., Kobayashi, K., Isler, M.P., McRee, D.E. & Ghadiri, M.R. (1998) "Cylindrical b-Sheet Peptide Assemblies." J. Amer. Chem. Soc. 120, 8949-8962.

Novel MAD Phasing Methods

MAD phased electron density

We are using synthetic means to add MAD phasing markers to proteins in collaboration with Phil Dawson.  We hope these techniques can be used in the future for  rapid and simple solutions to de novo crystallographic structures of proteins.

McRee, D.E.  (1992) "XtalView: A Visual Protein Crystallographic Software System for XII/XView."  J. Mol. Graphics 10, 44-47.
44. McRee, D.E. (1999) "XtalView/Xfit - A Versatile Program for Manipulating Atomic Coordinates and Electron Density."  J. Structural Biology125, 156-165.

The figures for this page were made with Molecular Images software. 

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