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The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998

Principles of Protein Structure for Chemical Recognition, Complementarity, and Catalysis

E.D. Getzoff, M. Aoyagi, A.S. Arvai, R.M. Brudler, C.M. Bruns, B.R. Crane, K.T. Forest, U.K. Genick, T.P.K. Lo, S.E. Mylvaganam, J.L. Pellequer, M.E. Pique, R.J. Rosenfeld, M.E. Stroupe, M.M. Thayer, M.J. Thompson

We study the structural chemistry underlying protein molecular recognition, complementarity, and catalysis at the atomic level. In these problem-directed studies, we use molecular biology, biochemistry, x-ray crystallography, and spectroscopy together with computational and computer graphics analysis to characterize the structural chemistry and function of proteins. We then test our understanding by protein design. State-of-the-art technologies we use include multiwavelength anomalous diffraction, time-resolved Laue crystallography, femtosecond and nanosecond laser initiation, and rapid spectroscopic characterization. Taken together, these methods enable us to provide a detailed understanding of how proteins work, rather than simply supply a description of protein structures.

Biocatalysis of Sulfur Transformations

Sulfite and nitrite reductases catalyze fundamental chemical transformations for biogeochemical cycling of sulfur and nitrogen. We used multiwavelength anomalous diffraction of the native siroheme and Fe4S4 cluster cofactors to solve the atomic resolution structure of sulfite reductase hemoprotein, which catalyzes the concerted 6-electron reductions of sulfite to sulfide and nitrite to ammonia. We determined 12 key high-resolution structures of sulfite reductase hemoprotein that characterize its active center in 3 different states of oxidation and its interactions with substrates, inhibitors, intermediates, and products. Coupled crystallographic and spectroscopic studies revealed the mechanism for heme activation via reduction-gated exogenous ligand exchange and provided descriptions of the intermediates at each step along the complex reaction pathway (Fig. 1).

Oxygen Recognition and Reactive Oxygen Catalysis

Ongoing research in the Getzoff and Tainer laboratories aims to understand the unique structural metallobiochemistry of nitric oxide synthase (NOS) and superoxide dismutase (SOD), which produce and regulate reactive oxygen species.

Cu,Zn SOD, which dismutes superoxide anions into oxygen and hydrogen peroxide, is a master eukaryotic regulator of reactive oxygen species, because most free radicals are scavenged by dioxygen to superoxide. Biochemically, eukaryotic Cu,Zn SODs are remarkable for their unusually great subunit and dimer stability, faster-than-diffusion attraction of substrate, exquisite specificity, and efficient catalysis. Biologically, SODs are important for their antioxidant, antiinflammatory, and antiaging properties. Moreover, by controlling levels of superoxide available for rapid reaction with nitric oxide to form peroxynitrite, SODs also regulate nitric oxide, an important biological signal and defensive cytotoxin (see following). Medically, genetic mutations in human Cu,Zn SOD cause the fatal degenerative disease of motor neurons termed amyotrophic lateral sclerosis or Lou Gehrig disease.

Our new structures of bacterial Cu,Zn SODs from symbionts and pathogens provide the potential for drug design. Whereas the fold and active-site geometry of bacterial Cu,Zn SODs match those of eukaryotic SODs, the elements recruited to form the dimer interface and the active-site channel are strikingly different. Our structures and redesign of SOD aim to elucidate the structural metallobiochemistry and structure-function relationships of the enzyme.

Our new research on the structural and chemical biology of NOS, which regulates the synthesis and thereby the biological activity of nitric oxide, complements our studies on SOD. Nitric oxide functions at low concentrations as a diffusible, biological messenger for neurotransmission, long-term potentiation, platelet aggregation, and regulation of blood pressure. At higher concentrations, nitric oxide acts as a cytotoxic agent for defense against tumor cells and intracellular parasites.

Each NOS subunit is divided into 2 domains joined by a calmodulin-binding hinge region: (1) an oxygenase domain with binding sites for heme, tetrahydrobiopterin, and substrate that forms the catalytic center for production of nitric oxide and (2) a reductase domain with binding sites for NADPH, FAD, and FMN that supplies electrons to the oxygenase domain. We crystallized both domains and determined refined structures for the oxygenase domain, in monomeric and dimeric forms, and in complex with cofactors, substrate, and inhibitors. Our structures revealed a novel protein fold and characterized the roles of the cofactors, and of the l-arginine substrate itself, in the 2 steps of the reaction mechanism. First, l-arginine is oxidized to N-hydroxy-l-arginine by a monooxygenase-like reaction; then this intermediate is oxidized to form citrulline and nitric oxide by an unprecedented reaction.

Chemical Biology of PAS Domains of Sensors and Clock Proteins

We aim to characterize the structural and chemical biology of PER-ARNT-SIM (PAS) domains (Fig. 2). These domains were originally identified as sequence repeats in the Drosophila clock protein PER and the basic helix-loop-helix--containing transcription factors aryl hydrocarbon receptor nuclear translocator (ARNT) in mammals and single-minded (SIM) in flies.

PAS domain sequences, which mediate protein-protein interaction and in some cases bind small ligands, also occur in sensor kinase proteins, the phytochrome photoreceptors of plants, and the newly discovered eukaryotic clock proteins, including the clock proteins that clearly couple photoreception to circadian rhythms. PAS domain sequences show similarities to photoactive yellow protein, a bacterial blue-light photosensor whose structural molecular biology has been under study. The aim of research funded by the Skaggs Institute is to use our 0.83-Å resolution structure of photoactive yellow protein to determine structures for PAS domains and to characterize the roles of these domains in signal transduction.

Coupled Crystallographic and Spectroscopic Analysis of Protein Mechanisms

The purpose of our project on coupled crystallographic and spectroscopic analysis of protein mechanisms is to establish new state-of-the-art technology at The Scripps Research Institute and provide leadership in a key area of chemical structural biology. The laser and spectroscopy facility that we established in this laboratory enables us to initiate and spectroscopically monitor reactions in protein crystals on a timescale of nanoseconds. This facility provides the opportunity for detailed characterization of the many interesting transient structural states of proteins responsible for catalysis and signaling, which typically have life-times shorter than milliseconds. Rapid monitoring of spectroscopic properties in protein crystals allows determination of the precise oxidation state of metal centers, specific assays of function in the crystalline state, and the ability to establish conditions that allow sufficient accumulation of transient intermediates for structure determination by time-resolved crystallography.


Crane, B., Siegel, L., Getzoff, E.D. Probing the catalytic mechanism of sulfite reductase by x-ray crystallography: Structure of the Eschericia 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., D.K., Getzoff, E.D., Stuehr, D.J., 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.



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