News and Publications

Principles of Protein Structure for Recognition, Interaction, Catalysis, and Design

E.D. Getzoff, M. Aoyagi, A.S. Arvai , D.P. Barondeau, S.L. Bernstein, R.M. Brudler, C.M. Bruns, I.L. Canestrelli, B.R. Crane, T.H. Cross, C.L. Fisher, K.T. Forest, U.K. Genick, F.R. Henderson, C.K. Koike, T.P.K. Lo, S.E. Mylvaganam, J.L. Pellequer, M.E. Pique, R.J. Rosenfeld, M.E. Stroup, M.M. Thayer, M.J. Thompson, T.T. Woo

We study functionally important protein conformational changes pertinent to protein recognition, interaction, and catalysis to provide a detailed understanding of how proteins work. Major research areas include protein photosensing, enzymatic control of reactive oxygen species, the coupling of metal-site chemistry and electron transfer for catalysis, and metalloprotein design. State-of-the-art technologies we use include multiwavelength anomalous diffraction; time-resolved Laue crystallography; freeze-trapping; femtosecond and nanosecond laser initiation; rapid or simultaneous spectroscopic characterization to monitor conformational states; computational and computer graphics analysis; and a protein-design cycle that integrates experimental results from x-ray crystallography, molecular biology, biochemistry, and spectroscopy.

PHOTOACTIVE YELLOW PROTEIN

To understand how a chromophore and a protein interact to sense light and send a biological signal, we determined structures for photocycle intermediates of photoactive yellow protein, a bacterial blue-light photosensor. With millisecond time-resolved Laue crystallography, we showed how trans-to-cis isomerization exposes the buried cinnamoyl chromophore to solvent and forms the likely signaling intermediate. Freeze-trapping followed by light activation produced high-resolution structures (Fig. 1) of the dark (0.82 Å) state and an early (0.85 Å) intermediate in the photocycle. Before significant movement of its aromatic ring can occur, the chromophore flips its thioester linkage with the protein to store energy from the incoming photon. Sequence similarities suggest that the photoactive yellow protein fold is the structural prototype for the superfamily of PAS (Per-Arnt-Sim) domains found in diverse biological sensors and in the clock proteins that control circadian rhythms.

SULFITE REDUCTASE

To elucidate how sulfite reductase catalyzes the 6-electron reductions of sulfite and nitrite required for the biogeochemical cycling of sulfur and nitrogen, we determined 12 key high-resolution structures of the catalytic hemoprotein subunit. These structures characterize the cysteine-linked siroheme and Fe₄S₄ cluster of the active center in 3 different states of oxidation and show interactions of the enzyme with substrates, inhibitors, intermediates, and products.

Reduction potential and reactivity of Fe₄S₄ are tuned by cofactor coupling, solvent accessibility, and protein hydrogen bonding. Our coupled crystallographic and spectroscopic studies revealed heme activation via reduction-gated exogenous ligand exchange and distinguished intermediates at each step along the complex reaction pathway. Siroheme binding by the sulfite substrate via sulfur provides unique placement of 1 oxygen atom (vs all other ligands studied), induces a spin transition in the siroheme iron, flips an active-site arginine, and orders surrounding active-center loops. Hydrogen bonds supplied by active-site arginine and lysine residues facilitate charge transfer into the substrate from the electron-rich cofactors, activate sulfur-oxygen bonds for reductive cleavage, and provide potential proton sources to form favorable aquo leaving groups.

SUPEROXIDE DISMUTASES AND NITRIC OXIDE SYNTHASES

Cu,Zn superoxide dismutases and nitric oxide synthases are metalloenzymes that control reactive oxygen in cells. By dismuting superoxide to oxygen and hydrogen peroxide, superoxide dismutases act as master regulators for intracellular radicals and reactive oxygen species. Our structures of superoxide dismutases from mammals and from bacterial symbionts and pathogens not only defined the common structural basis for the activity and stability of these enzymes but also revealed striking differences in the dimer interface, active-site channel, and disulfide bond. We determined the structures of oxidized, reduced, inhibitor-bound, and mutant mammalian superoxide dismutases to characterize the catalytic mechanism. On the basis of our results, we suggest a new hypothesis for the mechanism by which single-site mutations in human superoxide dismutase cause the fatal neurodegenerative disease amyotrophic lateral sclerosis.

Nitric oxide synthases oxidize arginine to synthesize the cellular signal and defensive cytotoxin nitric oxide. This synthesis requires interactions between the catalytic, heme-containing oxygenase domain and the electron-supplying reductase domain of the enzyme. Our crystallographic structures of the cytokine-inducible oxygenase domain revealed a novel fold and heme environment for stabilizing activated oxygen intermediates and showed how dimerization plus binding of substrate arginine and cofactor tetrahydrobiopterin create the catalytic center for synthesis of nitric oxide. Ongoing determination of the structure of the reductase domain revealed the fold of this domain and suggested a path for electron transfer to the oxygenase domain.

METALLOPROTEIN DESIGN

As members of the TSRI Metalloprotein Structure and Design Group, we examine the structural basis for the binding and activity of biologically important metal ions in proteins. We apply structure-based design algorithms to transplant metal-ion site templates from metalloproteins of known structure into 3 different protein scaffolds: antibodies, green fluorescent protein, and photoactive yellow protein.

Our antibody and antibody-protein complex structures are a powerful system for probing protein recognition, interaction, and catalysis, as well as the design of metal sites. Our free and antigen-bound structures of an antibody to cytochrome c revealed key roles for water at the antibody-antigen interface, showed propagation of local conformational changes upon binding, and indicated how distinct binding specificities can be achieved despite limited sequence differences. Our design, construction, and characterization of catalytic and metal-binding antibodies, in collaboration with V. Roberts, Department of Molecular Biology, and S. Benkovic, Pennsylvania State University, test and expand our understanding of metal-binding sites in proteins.

For metal-site design in green fluorescent protein and photoactive yellow protein, the naturally occurring chromophores (Fig. 2) allow spectroscopic monitoring and the development of metal-ion biosensors. These different systems test the roles of the protein framework in determining metal-site geometry and providing the environment needed for the affinity, specificity, and activity of metal-binding sites in proteins.

PUBLICATIONS

Crane, B., Siegel, L., Getzoff, E.D. Probing the catalytic mechanism of sulfite reductase by x-ray crystallography: Structure of the Escherichia coli hemoprotein in complex with substrates, inhibitors, intermediates and products. Biochemistry 36:12120, 1997.

Crane, B., Siegel, L., Getzoff, E.D. Structures of the siroheme and Fe₄S₄-containing active center of sulfite reductase in different states of oxidation: Heme activation via reduction-gated exogenous ligand exchange. Biochemistry 36:12101, 1997.

Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C., Ghosh, D.K., Getzoff, E.D., Stuehr, D.J., Tainer, J.A. The structure of nitric oxide synthase oxygenase domain and inhibitor complexes. Science 278:425, 1997.

Crane, B.R., Arvai, A.S., Ghosh, D.K., Wu, C., Getzoff, E.D., Stuehr, D., Tainer, J.A. Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 279:2121, 1998.

Devanathan, S., Genick, U.K., Canestrelli, I.L., Meyer, T.E., Cusanovich, M.A., Getzoff, E.D., Tollin, G. New insights into the photocycle of Ecotothiorhodospira halophila photoactive yellow protein: Photorecovery of the long-lived photobleached intermediate of the Met100Ala mutant. Biochemistry, in press.

Fisher, C.L., Cabelli, D.E., Hallewell, R.A., Beroza, P., Lo, T.P., Getzoff, E.D., Tainer, J.A. Computational, pulse-radiolytic, and structural investigations of lysine-136 and its role in the electrostatic triad of human Cu,Zn superoxide dismutase. Proteins 29:103, 1997.

Genick, U.K., Soltis, S.M., Kuhn, P., Canestrelli, I.L., Getzoff, E.D. Structure at 0.85-Å resolution of an early protein photocycle intermediate. Nature 392:206, 1998.

Mylvaganam, S.E., Paterson, Y., Getzoff, E.D. Structural basis for the binding of an anti-cytochrome c antibody to its antigen: Crystal structures of FabE8-cytochrome c complex to 1.8-Å resolution and FabE8 to 2.26-Å resolution. J. Mol. Biol., in press.

Pellequer, J.L., Wager-Smith, K.A., Kay, S.A., Getzoff, E.D. Photoactive yellow protein: A structural prototype for the three-dimensional fold of the PAS domain superfamily. Proc. Natl. Acad. Sci. U.S.A., in press.